Compositions and methods for treating diseases associated with an imprinting defect

ABSTRACT

The invention provides methods for activating a repressed allele within an imprinting control region, thereby treating an imprinting associated disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 16/631,762, filed Jan. 16, 2020, which is the U.S. National Stage application, pursuant to 35 U.S.C. § 371, of PCT International Application No. PCT/US2018/042876, filed Jul. 19, 2018, designating the United States and published in English, which claims the benefit of and priority to U.S. Provisional Application No. 62/534,532, filed Jul. 19, 2017, the entire contents of each of which are incorporated by reference herein.

SEQUENCE LISTING

The present application contains a Sequence Listing which has been submitted electronically in XML format following conversion from the originally filed TXT format.

The contents of the electronic XML Sequence Listing, (Date of creation: Jun. 26, 2023; Size: 64,726 bytes; Name: 167705-014106USDIV-SL.xml), and the original TXT format, is herein incorporated by reference in its entirety.

BACKGROUND

Sperm and oocytes are generated from primordial germ cells through distinct processes. Consequently, their genomes are packaged differently with distinct epigenetic landscapes. Following fertilization, paternal chromatin releases protamines and is repackaged with maternally-stored histones that are devoid of most histone modifications, while maternal chromatin harbors various histone modifications inherited from oocytes. The different processes of parental chromatin formation result in parental epigenetic asymmetry in zygotes, which becomes largely equalized during subsequent development with the exception of certain genomic loci, including imprinting control regions (ICRs). Errors in genomic imprinting can cause severe disorders and profound developmental defects, including, for example, Beckwith-Wiedemann, Angleman, and Prader-Willi syndromes, that lead to lifelong health problems. There is a significant need for improved therapies for the treatment of imprinting associated disorders.

SUMMARY

The invention provides methods for activating a repressed allele within an imprinting control region, thereby treating an imprinting associated disorder.

In one aspect, the invention provides a method of activating a histone H3 lysine 27 trimethylation (H3K27me3) repressed allele within an imprinting control region of a cell, the method comprising contacting the cell with an agent that inhibits histone H3 lysine 27 trimethylation, thereby activating the H3K27me3-repressed allele. In one embodiment, the agent is an inhibitor of the H3K27 methyltransferase. In another embodiment, the H3K27 methyltransferase is selected from the group consisting of EZH1, EZH2, PRC2, PRC2-Ezh1, or PRC2-Ezh2. In another embodiment, the agent is a small compound, polypeptide, or polynucleotide. In another embodiment, the agent is selected from the group consisting of tazemetostat, DZNep, GSK373, GSK126, El1, Epz005687, CPI-169.

In another aspect, the invention provides a method of activating a H3K27me3 repressed allele within an imprinting control region of a cell, the method comprising contacting the cell with an agent that selectively removes trimethylation at lysine 27 of histone 3, thereby activating the H3K27me3 repressed allele. In one embodiment, the agent is an H3K27me3-specific demethylase. In another embodiment, the agent is lysine-specific demethylase 6A (KDM6A), KDM6B, or KDM6C. In yet another embodiment, the cell is a mammalian cell in vitro or in vivo. In yet another embodiment, the cell is present in a mammal undergoing pre- or post-natal development.

In another aspect, the invention provides a method of treating a subject having a disorder associated with H3K27me3-dependent imprinting, the method comprising administering to the subject an agent that inhibits histone H3 lysine 27 trimethylation, thereby treating the disorder.

In another aspect, the invention provides a method of treating a subject having a disorder associated with H3K27me3-dependent imprinting, the method comprising administering to the subject an agent that selectively removes trimethylation at lysine 27, thereby treating the disorder.

In various embodiments, the disorder is associated with a mutation in a gene of Table 1 or selected from the group consisting of Adamts2, Bbx, BC049762, Bmp7, C430002E04Rik, E2f3, Enc1, Epas1, Etv6, Fam198b, G730013B05Rik, Gab1, Gramd1b, Mbnl2, Otx2, Otx2os1, Phf17, Rbms1, Rbp2, Runx1, Sfmbt2, Sh3gl3, Slc38a1, Slc38a2, Slc38a4, Smoc1, Sox21, and Tle3.

In various embodiments, the disorder is associated with a mutation in a gene selected from the group consisting of Sfmbt2, Bbx, C430002E04Rik, Phf17, Slc38a4, Gramd1b, Tle3, E2f3, Smoc1, Sox21, Slc38a1, Runx1, Bmp7, Rnc1, Fam198b, Rbms1, Zrsr1, Impact, and Fkbp6. In still other embodiments, the disorder is associated with a mutation in a gene selected from the group consisting of Sfmbt2, Gab1, Slc38a4, and Phf17. In still other embodiments, the disorder is associated with a mutation in a gene selected from the group consisting of Ev6, 17001125H03Rik, Smoc1, and Bmp7. In still other embodiments, the disorder is associated with a mutation in a gene selected from the group consisting of Gab1, Phf17, Sfmbt2, Slc38a4, or Smoc1. In still other embodiments, the disorder is microphthalmia with limb anomalies (MLA) associated with a mutation in Smoc1. In still other embodiments, the disorder is associated with limb development associated with a mutation in Smoc1. In still other embodiments, the disorder is associated with a placental defect associated with a mutation in Gab1 or Sfmbt2. In still other embodiments, one parental allele comprises a mutation and the other parental allele is a wild-type allele.

In another aspect, the invention provides a method of identifying a gene comprising H3K27me3-dependent imprinting, the method comprising analyzing chromatin derived from a biological sample for the presence of an H3K27me3 modification and identifying a gene having said modification.

In another aspect, the invention provides a method for characterizing H3K27me3-dependent imprinting in a sample, the method comprising analyzing chromatin derived from the sample for the presence of an H3K27me3 modification relative to a reference sample, thereby characterizing H3K27me3-dependent imprinting in the sample. In one embodiment, the sample is obtained from an embryo. In another embodiment, an increase or decrease in imprinting relative to the reference is associated with a developmental disorder. In particular embodiments,

the imprinting is in a gene selected from the group consisting of Adamts2, Bbx, BC049762, Bmp7, C430002E04Rik, E2f3, Enc1, Epas1, Etv6, Fam198b, G730013B5Rik, Gab1, Gramd1b, Mbnl2, Otx2, Otx2os1, Phf17, Rbms1, Rbp2, Runx1, Sfmbt2, Sh3gl3, Slc38a1, Slc38a2, Slc38a4, Smoc1, Sox21, and Tle3. In still other embodiments, the imprinting is in a gene selected from the group consisting of Sfmbt2, Bbx, C430002E04Rik, Phf17, Slc38a4, Gramd1b, Tle3, E2f3, Smoc1, Sox21, Slc38a1, Runx1, Bmp7, Rnc1, Fam198b, Rbms1, Zrsr1, Impact, and Fkbp6. In still other embodiments, the imprinting is in a gene selected from the group Sfmbt2, Gab1, Slc38a4, and Phf17. In other embodiments, the imprinting is in a gene selected from the group Etv6, 17001125H03Rik, Smoc1, and Bmp7.

In another aspect, the invention provides a method for increasing histone H3 lysine 27 trimethylation (H3K27me3) within an imprinting control region of a hybrid cell, the method comprising contacting a donor mammalian cell, donor nucleus, recipient mammalian oocyte, hybrid cell, with an agent that increases histone H3 lysine 27 trimethylation (H3K27me3), thereby increasing histone H3 lysine 27 trimethylation (H3K27me3) within an imprinting control region of a hybrid cell. In one embodiment, the agent is an mRNA encoding an H3K27 methyltransferase.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By “EZH1 polypeptide” (histone-lysine N-methyltransferase EZH1) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP_001982, or a fragment thereof, and having methyltransferase activity. An exemplary H3K27 methyltransferase amino acid sequence is provided below (SEQ ID NO: 1):

1 meipnpptsk citywkrkvk seymrlrqlk rlqanmgaka lyvanfakvq ektqilneew  61 kklrvqpvqs mkpvsghpfl kkctiesifp gfasqhmlmr slntvalvpi myswsplqqn  121 fmvedetvlc nipymgdevk eedetfieel innydgkvhg eeemipgsvl isdavflelv 181 dalnqysdee eeghndtsdg kqddskedlp vtrkrkrhai egnkksskkq fpndmifsai 241 asmfpengvp ddmkeryrel temsdpnalp pqctpnidgp naksvqreqs lhsfhtlfcr 301 rcfkydcflh pfhatpnvyk rknkeikiep epcgtdcfll legakeyaml hnprskcsgr 361 rrrrhhivsa scsnasasav aetkegdsdr dtgndwasss seansrcqtp tkqkaspapp 421 qlcvveapse pvewtgaees lfrvfhgtyf nnfcsiarll gtktckqvfq favkeslilk 481 lptdelmnps qkkkrkhrlw aahcrkiqlk kdnsstqvyn yqpcdhpdrp cdstcpcimt 541 qnfcekfcqc npdcqnrfpg crcktqentk qcpcylavre cdpdlcltcg asehwdckvv  601 sckncsiqrg lkkhlllaps dvagwgtfik esvqknefis eycgelisqd eadrrgkvyd  661 kymssflfnl nndfvvdatr kgnkirfanh svnpncyakv vmvngdhrig ifakraiqag 721 eelffdyrys qadalkyvgi eretdvl 

By “EZH1 polynucleotide” is meant a nucleic acid molecule encoding the EZH1 polypeptide. An exemplary EZH1 polynucleotide sequence is provided at NM_001991.4 and reproduced below (SEQ ID NO: 2):

1 aggaggcgcg gggcggggca cggcgcaggg gtggggccgc ggcgcgcatg cgtcctagca 61 gcgggacccg cggctcggga tggaggctgg acacctgttc tgctgttgtg tcctgccatt 121 ctcctgaaga acagaggcac actgtaaaac ccaacacttc cccttgcatt ctataagatt 181 acagcaagat ggaaatacca aatcccccta cctccaaatg tatcacttac tggaaaagaa 241 aagtgaaatc tgaatacatg cgacttcgac aacttaaacg gcttcaggca aatatgggtg 301 caaaggcttt gtatgtggca aattttgcaa aggttcaaga aaaaacccag atcctcaatg 361 aagaatggaa gaagcttcgt gtccaacctg ttcagtcaat gaagcctgtg agtggacacc 421 cttttctcaa aaagtgtacc atagagagca ttttcccggg atttgcaagc caacatatgt 481 taatgaggtc actgaacaca gttgcattgg ttcccatcat gtattcctgg tcccctctcc 541 aacagaactt tatggtagaa gatgagacgg ttttgtgcaa tattccctac atgggagatg 601 aagtgaaaga agaagatgag acttttattg aggagctgat caataactat gatgggaaag 661 tccatggtga agaagagatg atccctggat ccgttctgat tagtgatgct gtttttctgg 721 agttggtcga tgccctgaat cagtactcag atgaggagga ggaagggcac aatgacacct 781 cagatggaaa gcaggatgac agcaaagaag atctgccagt aacaagaaag agaaagcgac 841 atgctattga aggcaacaaa aagagttcca agaaacagtt cccaaatgac atgatcttca 901 gtgcaattgc ctcaatgttc cctgagaatg gtgtcccaga tgacatgaag gagaggtatc 961 gagaactaac agagatgtca gaccccaatg cacttccccc tcagtgcaca cccaacatcg 1021 atggccccaa tgccaagtct gtgcagcggg agcaatctct gcactccttc cacacacttt 1081 tttgccggcg ctgctttaaa tacgactgct tccttcaccc ttttcatgcc acccctaatg 1141 tatataaacg caagaataaa gaaatcaaga ttgaaccaga accatgtggc acagactgct 1201 tccttttgct ggaaggagca aaggagtatg ccatgctcca caacccccgc tccaagtgct 1261 ctggtcgtcg ccggagaagg caccacatag tcagtgcttc ctgctccaat gcctcagcct 1321 ctgctgtggc tgagactaaa gaaggagaca gtgacaggga cacaggcaat gactgggcct 1381 ccagttcttc agaggctaac tctcgctgtc agactcccac aaaacagaag gctagtccag 1441 ccccacctca actctgcgta gtggaagcac cctcggagcc tgtggaatgg actggggctg 1501 aagaatctct ttttcgagtc ttccatggca cctacttcaa caacttctgt tcaatagcca 1561 ggcttctggg gaccaagacg tgcaagcagg tctttcagtt tgcagtcaaa gaatcactta 1621 tcctgaagct gccaacagat gagctcatga acccctcaca gaagaagaaa agaaagcaca 1681 gattgtgggc tgcacactgc aggaagattc agctgaagaa agataactct tccacacaag 1741 tgtacaacta ccaaccctgc gaccacccag accgcccctg tgacagcacc tgcccctgca 1801 tcatgactca gaatttctgt gagaagttct gccagtgcaa cccagactgt cagaatcgtt 1861 tccctggctg tcgctgtaag acccagtgca ataccaagca atgtccttgc tatctggcag 1921 tgcgagaatg tgaccctgac ctgtgtctca cctgtggggc ctcagagcac tgggactgca 1981 aggtggtttc ctgtaaaaac tgcagcatcc agcgtggact taagaagcac ctgctgctgg 2041 ccccctctga tgtggccgga tggggcacct tcataaagga gtctgtgcag aagaacgaat 2101 tcatttctga atactgtggt gagctcatct ctcaggatga ggctgatcga cgcggaaagg 2161 tctatgacaa atacatgtcc agcttcctct tcaacctcaa taatgatttt gtagtggatg 2221 ctactcggaa aggaaacaaa attcgatttg caaatcattc agtgaatccc aactgttatg 2281 ccaaagtggt catggtgaat ggagaccatc ggattgggat ctttgccaag agggcaattc 2341 aagctggcga agagctcttc tttgattaca ggtacagcca agctgatgct ctcaagtacg 2401 tggggatcga gagggagacc gacgtccttt agccctccca ggccccacgg cagcacttat 2461 ggtagcggca ctgtcttggc tttcgtgctc acaccactgc tgctcgagtc tcctgcactg 2521 tgtctcccac actgagaaac cccccaaccc actccctctg tagtgaggcc tctgccatgt 2581 ccagagggca caaaactgtc tcaatgagag gggagacaga ggcagctagg gcttggtctc 2641 ccaggacaga gagttacaga aatgggagac tgtttctctg gcctcagaag aagcgagcac 2701 aggctggggt ggatgactta tgcgtgattt cgtgtcggct ccccaggctg tggcctcagg 2761 aatcaactta ggcagttccc aacaagcgct agcctgtaat tgtagctttc cacatcaaga 2821 gtccttatgt tattgggatg caggcaaacc tctgtggtcc taagacctgg agaggacagg 2881 ctaagtgaag tgtggtccct ggagcctaca agtggtctgg gttagaggcg agcctggcag 2941 gcagcacaga ctgaactcag aggtagacag gtcaccttac tacctcctcc ctcgtggcag 3001 ggctcaaact gaaagagtgt gggttctaag tacaggcatt caaggctggg ggaaggaaag 3061 ctacgccatc cttccttagc cagagaggga gaaccagcca gatgatagta gttaaactgc 3121 taagcttggg cccaggaggc tttgagaaag ccttctctgt gtactctgga gatagatgga 3181 gaagtgtttt cagattcctg ggaacagaca ccagtgctcc agctcctcca aagttctggc 3241 ttagcagctg caggcaagca ttatgctgct attgaagaag cattaggggt atgcctggca 3301 ggtgtgagca tcctggctcg ctggatttgt gggtgttttc aggccttcca ttccccatag 3361 aggcaaggcc caatggccag tgttgcttat cgcttcaggg taggtgggca caggcttgga 3421 ctagagagga gaaagattgg tgtaatctgc tttcctgtct gtagtgcctg ctgtttggaa 3481 agggtgagtt agaatatgtt ccaaggttgg tgaggggcta aattgcacgc gtttaggctg 3541 gcaccccgtg tgcagggcac actggcagag ggtatctgaa gtgggagaag aagcaggtag 3601 accacctgtc ccaggctgtg gtgccaccct ctctggcatt catgcagagc aaagcacttt 3661 aaccatttct tttaaaaggt ctatagattg gggtagagtt tggcctaagg tctctagggt 3721 ccctgcctaa atcccactcc tgagggaggg ggaagaagag agggtgggag attctcctcc 3781 agtcctgtct catctcctgg gagaggcaga cgagtgagtt tcacacagaa gaatttcatg 3841 tgaatggggc cagcaagagc tgccctgtgt ccatggtggg tgtgccgggc tggctgggaa 3901 caaggagcag tatgttgagt agaaagggtg tgggcgggta tagattggcc tgggagtgtt 3961 acagtaggga gcaggcttct cccttctttc tgggactcag agccccgctt cttcccactc 4021 cacttgttgt cccatgaagg aagaagtggg gttcctcctg acccagctgc ctcttacggt 4081 ttggtatggg acatgcacac acactcacat gctctcactc accacactgg agggcacaca 4141 cgtaccccgc acccagcaac tcctgacaga aagctcctcc cacccaaatg ggccaggccc 4201 cagcatgatc ctgaaatctg catccgccgt ggtttgtatt cattgtgcat atcagggata 4261 ccctcaagct ggactgtggg ttccaaatta ctcatagagg agaaaaccag agaaagatga 4321 agaggaggag ttaggtctat ttgaaatgcc aggggctcgc tgtgaggaat aggtgaaaaa 4381 aaacttttca ccagcctttg agagactaga ctgaccccac ccttccttca gtgagcagaa 4441 tcactgtggt cagtctcctg tcccagcttc agttcatgaa tactcctgtt cctccagttt 4501 cccatccttt gtccctgctg tcccccactt ttaaagatgg gtctcaaccc ctccccacca 4561 cgtcatgatg gatggggcaa ggtggtgggg actaggggag cctggtatac atgcggcttc 4621 attgccaata aatttcatgc actttaaagt cctgtggctt gtgacctctt aataaagtgt 4681 tagaatccaa aaaaaaa

By “EZH2 polypeptide” (histone-lysine N-methyltransferase EZH2) is meant a protein having at least about 85% amino acid identity to the sequence provided at UniProtKB/Swiss-Prot: Q15910.2, or a fragment thereof, and having methyltransferase activity. An exemplary H3K27 methyltransferase amino acid sequence is provided below (SEQ ID NO: 3):

  1 mgqtgkksek gpvcwrkrvk seymrlrqlk rfrradevks mfssnrqkil erteilngew  61 kqrriqpvhi ltsvsslrgt recsvtsdld fptqviplkt lnavasvpim yswsplqqnf 121 mvedetvlhn ipymgdevld qdgtfieeli knydgkvhgd recgfindei fvelvnalgq 181 yndddddddg ddpeereekq kdledhrddk esrpprkfps dkifeaissm fpdkgtaeel 241 kekykelteq qlpgalppec tpnidgpnak svqreqslhs fhtlfcrrcf kydcflhpfh 301 atpntykrkn tetaldnkpc gpqcyqhleg akefaaalta eriktppkrp ggrrrgrlpn 361 nssrpstpti nvleskdtds dreagtetgg enndkeeeek kdetssssea nsrcqtpikm 421 kpnieppenv ewsgaeasmf rvligtyydn fcaiarligt ktorqvyefr vkessiiapa 481 paedvdtppr kkkrkhrlwa ahcrkiqlkk dgssnhvyny qpcdhprqpc dsscpcviaq 541 nfcekfcqcs secqnrfpgc rckagentkq cpcylavrec dpdlcltcga adhwdsknvs 601 ckncsiqrgs kkhlllapsd vagwgifikd pvqknefise ycgeiisqde adrrgkvydk 661 ymcsflfnln ndfvvdatrk gnkirfanhs vnpncyakvm mvngdhrigi fakraiqtge 721 elffdyrysq adalkyvgie remeip

By “EZH2 polynucleotide” is meant a nucleic acid molecule encoding an EZH2 polypeptide. An exemplary EZH2 polynucleotide sequence is provided at NM_001203248.1 and is provided below (SEQ ID NO: 4):

1 ggcggcgctt gattgggctg ggggggccaa ataaaagcga tggcgattgg gctgccgcgt 61 ttggcgctcg gtccggtcgc gtccgacacc cggtgggact cagaaggcag tggagccccg 121 gcggcggcgg cggcggcgcg cgggggcgac gcgcgggaac aacgcgagtc ggcgcgcggg 181 acgaagaata atcatgggcc agactgggaa gaaatctgag aagggaccag tttgttggcg 241 gaagcgtgta aaatcagagt acatgcgact gagacagctc aagaggttca gacgagctga 301 tgaagtaaag agtatgttta gttccaatcg tcagaaaatt ttggaaagaa cggaaatctt 361 aaaccaagaa tggaaacagc gaaggataca gcctgtgcac atcctgactt cttgttcggt 421 gaccagtgac ttggattttc caacacaagt catcccatta aagactctga atgcagttgc 481 ttcagtaccc ataatgtatt cttggtctcc cctacagcag aattttatgg tggaagatga 541 aactgtttta cataacattc cttatatggg agatgaagtt ttagatcagg atggtacttt 601 cattgaagaa ctaataaaaa attatgatgg gaaagtacac ggggatagag aatgtgggtt 661 tataaatgat gaaatttttg tggagttggt gaatgccctt ggtcaatata atgatgatga 721 cgatgatgat gatggagacg atcctgaaga aagagaagaa aagcagaaag atctggagga 781 tcaccgagat gataaagaaa gccgcccacc tcggaaattt ccttctgata aaatttttga 841 agccatttcc tcaatgtttc cagataaggg cacagcagaa gaactaaagg aaaaatataa 901 agaactcacc gaacagcagc tcccaggcgc acttcctcct gaatgtaccc ccaacataga 961 tggaccaaat gctaaatctg ttcagagaga gcaaagctta cactcctttc atacgctttt 1021 ctgtaggcga tgttttaaat atgactgctt cctacatcct tttcatgcaa cacccaacac 1081 ttataagcgg aagaacacag aaacagctct agacaacaaa ccttgtggac cacagtgtta 1141 ccagcatttg gagggagcaa aggagtttgc tgctgctctc accgctgagc ggataaagac 1201 cccaccaaaa cgtccaggag gccgcagaag aggacggctt cccaataaca gtagcaggcc 1261 cagcaccccc accattaatg tgctggaatc aaaggataca gacagtgata gggaagcagg 1321 gactgaaacg gggggagaga acaatgataa agaagaagaa gagaagaaag atgaaacttc 1381 gagctcctct gaagcaaatt ctcggtgtca aacaccaata aagatgaagc caaatattga 1441 acctcctgag aatgtggagt ggagtggtgc tgaagcctca atgtttagag tcctcattgg 1501 cacttactat gacaatttct gtgccattgc taggttaatt gggaccaaaa catgtagaca 1561 ggtgtatgag tttagagtca aagaatctag catcatagct ccagctcccg ctgaggatgt 1621 ggatactcct ccaaggaaaa agaagaggaa acaccggttg tgggctgcac actgcagaaa 1681 gatacagctg aaaaaggacg gctcctctaa ccatgtttac aactatcaac cctgtgatca 1741 tccacggcag ccttgtgaca gttcgtgccc ttgtgtgata gcacaaaatt tttgtgaaaa 1801 gttttgtcaa tgtagttcag agtgtcaaaa ccgctttccg ggatgccgct gcaaagcaca 1861 gtgcaacacc aagcagtgcc cgtgctacct ggctgtccga gagtgtgacc ctgacctctg 1921 tcttacttgt ggagccgctg accattggga cagtaaaaat gtgtcctgca agaactgcag 1981 tattcagcgg ggctccaaaa agcatctatt gctggcacca tctgacgtgg caggctgggg 2041 gatttttatc aaagatcctg tgcagaaaaa tgaattcatc tcagaatact gtggagagat 2101 tatttctcaa gatgaagctg acagaagagg gaaagtgtat gataaataca tgtgcagctt 2161 tctgttcaac ttgaacaatg attttgtggt ggatgcaacc cgcaagggta acaaaattcg 2221 ttttgcaaat cattcggtaa atccaaactg ctatgcaaaa gttatgatgg ttaacggtga 2281 tcacaggata ggtatttttg ccaagagagc catccagact ggcgaagagc tgttttttga 2341 ttacagatac agccaggctg atgccctgaa gtatgtcggc atcgaaagag aaatggaaat 2401 cccttgacat ctgctacctc ctcccccctc ctctgaaaca gctgccttag cttcaggaac 2461 ctcgagtact gtgggcaatt tagaaaaaga acatgcagtt tgaaattctg aatttgcaaa 2521 gtactgtaag aataatttat agtaatgagt ttaaaaatca actttttatt gccttctcac 2581 cagctgcaaa gtgttttgta ccagtgaatt tttgcaataa tgcagtatgg tacatttttc 2641 aactttgaat aaagaatact tgaacttgtc cttgttgaat c

By “KDM6A polypeptide” (lysine-specific demethylase 6A, also referred to as histone demethylase UTX) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: 015550.2, or a fragment thereof, and having demethylase activity. An exemplary KDM6A amino acid sequence is provided below (SEQ ID NO: 5):

1 mkscgvslat aaaaaaafgd eekkmaagka sgeseeasps ltaeerealg gldsrlfgfv 61 rfhedgartk allgkavrcy eslilkaegk vesdffcqlg hfnllledyp kalsayqryy 121 slqsdywkna aflyglglvy fhynafqwai kafqevlyvd psfcrakeih lrlglmfkvn 181 tdyesslkhf qlalvdcnpc tlsnaeiqfh iahlyetqrk yhsakeayeq llqtenlsaq 241 vkatvlqqlg wmhhtvdllg dkatkesyai qylqkslead pnsgqswyfl grcyssigkv 301 qdafisyrqs idkseasadt wcsigvlyqq qnqpmdalqa yicavqldhg haaawmdlgt 361 lyescnqpqd aikcylnatr skscsntsal aarikylqaq lcnlpqgslq nktkllpsie 421 eawslpipae ltsrqgamnt aqqntsdnws gghavshppv qqqahswelt pqklqhleql 481 ranrnnlnpa qklmleqles qfvlmqqhqm rptgvaqvrs tgipngptad sslptnsvsg 541 qqpqlaltrv psvsqpgvrp acpgqplang pfsaghvpcs tsrtlgstdt ilignnhitg 601 sgsngnvpyl qrnaltlphn rtnltssaee pwknqlsnst qglhkgqssh sagpngerpl 661 sstgpsqhlq aagsgiqnqn ghptlpsnsv tqgaalnhls shtatsggqq gitltkeskp 721 sgniltvpet srhtgetpns tasveglpnh vhqmtadavc spshgdsksp gllssdnpql 781 sallmgkann nvgtgtcdkv nnihpavhtk tdnsvassps saistatpsp ksteqtttns 841 vtslnsphsg lhtingegme esqspmktdl llvnhkpspq iipsmsvsiy pssaevlkac 901 rnlgknglsn ssilldkopp prppsspypp lpkdklnppt psiylenkrd affpplhqfc 961 tnpnnpvtvi rglagalkld lglfstktlv eannehmvev rtqllqpade nwdptgtkki 1021 whcesnrsht tiakyaqyqa ssfqeslree nekrshhkdh sdsestssdn sgrrrkgpfk 1081 tikfgtnidl sddkkwklql heltklpafv rvvsagnlls hvghtilgmn tvqlymkvpg 1141 srtpghqenn nfcsvninig pgdcewfvvp egywgvlndf ceknnlnflm gswwpnledl 1201 yeanvpvyrf iqrpgdlvwi nagtvhwvga igwcnniawn vgpltacqyk laveryewnk 1261 lqsvksivpm vhlswnmarn ikvsdpklfe mikycllrtl kqcqtlreal iaagkeiiwh 1321 grtkeepahy csicevevfd llfvtnesns rktyivhcqd carktsgnle nfvvleqykm 1381 edlmqvydqf tlapplpsas s

By “KDM6A polynucleotide” is meant a nucleic acid molecule encoding a KDM6A polypeptide. An exemplary KDM6A polynucleotide sequence is provided at NM_001291415.1.

By “KDM6B polypeptide” (lysine-specific demethylase 6, also referred to as JmjC domain-containing protein 3) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: 015054.4, or a fragment thereof, and having demethylase activity. An exemplary KDM6B amino acid sequence is provided below (SEQ ID NO: 6):

1 mhravdppga raareafalg glscagawss cpphppprsa wlpggrcsas igqpplpapl 61 ppshgsssgh pskpyyapga ptprplhgkl eslhgcvqal lrepaqpglw eqlgqlyese 121 hdseeatrcy hsalryggsf aelgprigrl qqaqlwnfht gscqhrakvl ppleqvwnll 181 hlehkrnyga krggppvkra aeppvvqpvp paalsgpsge eglspggkrr rgenseqtgl 241 ppglplpppp lppppppppp pppplpglat sppfqltkpg lwstlhgdaw gperkgsapp 301 erqeqrhslp hpypypapay tahppghrlv paappgpgpr ppgaeshgcl patrppgsdl 361 resrvqrsrm dssvspaatt acvpyapsrp pglpgtttss ssssssntgl rgvepnpgip 421 gadhyqtpal evshhgrlgp sahssrkpfl gapaatphls lppgpssppp ppcprllrpp 481 pppawlkgpa craaredgei leelffgteg pprpappplp hregflgppa srfsvgtqds 541 htpptpptpt tsssnsnsgs hssspagpvs fppppylars idplprppsp aqnpqdpplv 601 pltlalppap psschqntsg sfrrpesprp rvsfpktpev gpgpppgpls kapqpvppgv 661 gelpargprl fdfpptpled qfeepaefki lpdglanimk mldesirkee eqqqheagva 721 pqpplkepfa slqspfptdt aptttapava vttttttttt ttatqeeekk pppalppppp 781 lakfpppsqp qpppppppsp asllkslasv legqkycyrg tgaavstrpg plpttqyspg 841 ppsgatalpp tsaapsaqgs pqpsassssq fstsggpwar errageepvp gpmtptqppp 901 plslpparse sevleeisra cetlvervgr satdpadpvd taepadsgte rllppaqake 961 eaggvaavsg sckrrqkehq kehrrhrrac kdsvgrrpre grakakakvp keksrrvlgn 1021 ldlqseeiqg reksrpdlgg askakpptap appsapapsa qptppsasvp gkkareeapg 1081 ppgvsradml klrslsegpp kelkirlikv esgdketfia seveerrlrm adltishcaa 1141 dvvrasrnak vkgkfresyl spaqsvkpki nteeklprek lnpptpsiyl eskrdafspv 1201 llqfctdprn pitvirglag slrlnlglfs tktlveasge htvevrtqvq qpsdenwdlt 1261 gtrqiwpces srshttiaky aqyqassfqe slqeekesed eeseepdstt gtppssapdp 1321 knhhiikfgt nidlsdakrw kpqlqellkl pafmrvtstg nmlshvghti lgmntvqlym 1381 kvpgsrtpgh qennnfcsvn inigpgdcew favhehywet isafcdrhgv dyltgswwpi 1441 lddlyasnip vyrfvqrpgd lvwinagtvh wvqatgwenn iawnvgplta yqyqlalery 1501 ewnevknvks ivpmihvswn vartvkisdp dlfkmikfcl lqsmkhcqvq reslvragkk 1561 iayqgrvkde payycnecdv evfnilfvts engsrntylv hcegcarrrs aglqgvvvle 1621 qyrteelaga ydaftlapas tsr

By “KDM6B polynucleotide” is meant a nucleic acid molecule encoding a KDM6B polypeptide. An exemplary KDM6B polynucleotide sequence is provided at NM 001080424.2 and reproduced below SE ID NO: 7):

1 ggcaacatgc cagccccgta gcactgccca ccccacccac tgtggtctgt tgtaccccac 61 tgctggggtg gtggttccaa tgagacaggg cacaccaaac tccatctggc tgttactgag 121 gcggagacac gggtgatgat tggctttctg gggagagagg aagtcctgtg attggccaga 181 tctctggagc ttgccgacgc ggtgtgagga cgctcccacg gaggccggaa ttggctgtga 241 aaggactgag gcagccatct gggggtagcg ggcactctta tcagagcggc tggagccgga 301 ccatcgtccc agagagctgg ggcagggggc cgtgcccaat ctccagggct cctggggcca 361 ctgctgacct ggctggatgc atcgggcagt ggaccctcca ggggcccgcg ctgcacggga 421 agcctttgcc cttgggggcc tgagctgtgc tggggcctgg agctcctgcc cgcctcatcc 481 ccctcctcgt agcgcatggc tgcctggagg cagatgctca gccagcattg ggcagccccc 541 gcttcctgct cccctacccc cttcacatgg cagtagttct gggcacccca gcaaaccata 601 ttatgctcca ggggcgccca ctccaagacc cctccatggg aagctggaat ccctgcatgg 661 ctgtgtgcag gcattgctcc gggagccagc ccagccaggg ctttgggaac agcttgggca 721 actgtacgag tcagagcacg atagtgagga ggccacacgc tgctaccaca gcgcccttcg 781 atacggagga agcttcgctg agctggggcc ccgcattggc cgactgcagc aggcccagct 841 ctggaacttt catactggct cctgccagca ccgagccaag gtcctgcccc cactggagca 901 agtgtggaac ttgctacacc ttgagcacaa acggaactat ggagccaagc ggggaggtcc 961 cccggtgaag cgagctgctg aacccccagt ggtgcagcct gtgcctcctg cagcactctc 1021 aggcccctca ggggaggagg gcctcagccc tggaggcaag cgaaggagag gctgcaactc 1081 tgaacagact ggccttcccc cagggctgcc actgcctcca ccaccattac caccaccacc 1141 accaccacca ccaccaccac caccacccct gcctggcctg gctaccagcc ccccatttca 1201 gctaaccaag ccagggctgt ggagtaccct gcatggagat gcctggggcc cagagcgcaa 1261 gggttcagca cccccagagc gccaggagca gcggcactcg ctgcctcacc catatccata 1321 cccagctcca gcgtacaccg cgcacccccc tggccaccgg ctggtcccgg ctgctccccc 1381 aggcccaggc ccccgccccc caggagcaga gagccatggc tgcctgcctg ccacccgtcc 1441 ccccggaagt gaccttagag agagcagagt tcagaggtcg cggatggact ccagcgtttc 1501 accagcagca accaccgcct gcgtgcctta cgccccttcc cggccccctg gcctccccgg 1561 caccaccacc agcagcagca gtagcagcag cagcaacact ggtctccggg gcgtggagcc 1621 gaacccaggc attcccggcg ctgaccatta ccaaactccc gcgctggagg tctctcacca 1681 tggccgcctg gggccctcgg cacacagcag tcggaaaccg ttcttggggg ctcccgctgc 1741 cactccccac ctatccctgc cacctggacc ttcctcaccc cctccacccc cctgtccccg 1801 cctcttacgc cccccaccac cccctgcctg gttgaagggt ccggcctgcc gggcagcccg 1861 agaggatgga gagatcttag aagagctctt ctttgggact gagggacccc cccgccctgc 1921 cccaccaccc ctcccccatc gcgagggctt cttggggcct ccggcctccc gcttttctgt 1981 gggcactcag gattctcaca cccctcccac tcccccaacc ccaaccacca gcagtagcaa 2041 cagcaacagt ggcagccaca gcagcagccc tgctgggcct gtgtcctttc ccccaccacc 2101 ctatctggcc agaagtatag acccccttcc ccggcctccc agcccagcac agaaccccca 2161 ggacccacct cttgtacccc tgactcttgc cctgcctcca gcccctcctt cctcctgcca 2221 ccaaaatacc tcaggaagct tcaggcgccc ggagagcccc cggcccaggg tctccttccc 2281 aaagaccccc gaggtggggc cggggccacc cccaggcccc ctgagtaaag ccccccagcc 2341 tgtgccgccc ggggttgggg agctgcctgc ccgaggccct cgactctttg attttccccc 2401 cactccgctg gaggaccagt ttgaggagcc agccgaattc aagatcctac ctgatgggct 2461 ggccaacatc atgaagatgc tggacgaatc cattcgcaag gaagaggaac agcaacaaca 2521 cgaagcaggc gtggcccccc aacccccgct gaaggagccc tttgcatctc tgcagtctcc 2581 tttccccacc gacacagccc ccaccactac tgctcctgct gtcgccgtca ccaccaccac 2641 caccaccacc accaccacca cggccaccca ggaagaggag aagaagccac caccagccct 2701 accaccacca ccgcctctag ccaagttccc tccaccctct cagccacagc caccaccacc 2761 cccacccccc agcccggcca gcctgctcaa atccttggcc tccgtgctgg agggacaaaa 2821 gtactgttat cgggggactg gagcagctgt ttccacccgg cctgggccct tgcccaccac 2881 tcagtattcc cctggccccc catcaggtgc taccgccctg ccgcccacct cagcggcccc 2941 tagcgcccag ggctccccac agccctctgc ttcctcgtca tctcagttct ctacctcagg 3001 cgggccctgg gcccgggagc gcagggcggg cgaagagcca gtcccgggcc ccatgacccc 3061 cacccaaccg cccccacccc tatctctgcc ccctgctcgc tctgagtctg aggtgctaga 3121 agagatcagc cgggcttgcg agacccttgt ggagcgggtg ggccggagtg ccactgaccc 3181 agccgaccca gtggacacag cagagccagc ggacagtggg actgagcgac tgctgccccc 3241 cgcacaggcc aaggaggagg ctggcggggt ggcggcagtg tcaggcagct gtaagcggcg 3301 acagaaggag catcagaagg agcatcggcg gcacaggcgg gcctgtaagg acagtgtggg 3361 tcgtcggccc cgtgagggca gggcaaaggc caaggccaag gtccccaaag aaaagagccg 3421 ccgggtgctg gggaacctgg acctgcagag cgaggagatc cagggtcgtg agaagtcccg 3481 gcccgatctt ggcggggcct ccaaggccaa gccacccaca gctccagccc ctccatcagc 3541 tcctgcacct tctgcccagc ccacaccccc gtcagcctct gtccctggaa agaaggctcg 3601 ggaggaagcc ccagggccac cgggtgtcag ccgggccgac atgctgaagc tgcgctcact 3661 tagtgagggg ccccccaagg agctgaagat ccggctcatc aaggtagaga gtggtgacaa 3721 ggagaccttt atcgcctctg aggtggaaga gcggcggctg cgcatggcag acctcaccat 3781 cagccactgt gctgctgacg tcgtgcgcgc cagcaggaat gccaaggtga aagggaagtt 3841 tcgagagtcc tacctttccc ctgcccagtc tgtgaaaccg aagatcaaca ctgaggagaa 3901 gctgccccgg gaaaaactca acccccctac acccagcatc tatctggaga gcaaacggga 3961 tgccttctca cctgtcctgc tgcagttctg tacagaccct cgaaatccca tcacagtgat 4021 ccggggcctg gcgggctccc tgcggctcaa cttgggcctc ttctccacca agaccctggt 4081 ggaagcgagt ggcgaacaca ccgtggaagt tcgcacccag gtgcagcagc cctcagatga 4141 gaactgggat ctgacaggca ctcggcagat ctggccttgt gagagctccc gttcccacac 4201 caccattgcc aagtacgcac agtaccaggc ctcatccttc caggagtctc tgcaggagga 4261 gaaggagagt gaggatgagg agtcagagga gccagacagc accactggaa cccctcctag 4321 cagcgcacca gacccgaaga accatcacat catcaagttt ggcaccaaca tcgacttgtc 4381 tgatgctaag cggtggaagc cccagctgca ggagctgctg aagctgcccg ccttcatgcg 4441 ggtaacatcc acgggcaaca tgctgagcca cgtgggccac accatcctgg gcatgaacac 4501 ggtgcagctg tacatgaagg tgcccggcag ccgaacgcca ggccaccagg agaataacaa 4561 cttctgctcc gtcaacatca acattggccc aggcgactgc gagtggttcg cggtgcacga 4621 gcactactgg gagaccatca gcgctttctg tgatcggcac ggcgtggact acttgacggg 4681 ttcctggtgg ccaatcctgg atgatctcta tgcatccaat attcctgtgt accgcttcgt 4741 gcagcgaccc ggagacctcg tgtggattaa tgcggggact gtgcactggg tgcaggccac 4801 cggctggtgc aacaacattg cctggaacgt ggggcccctc accgcctatc agtaccagct 4861 ggccctggaa cgatacgagt ggaatgaggt gaagaacgtc aaatccatcg tgcccatgat 4921 tcacgtgtca tggaacgtgg ctcgcacggt caaaatcagc gaccccgact tgttcaagat 4981 gatcaagttc tgcctgctgc agtccatgaa gcactgccag gtgcaacgcg agagcctggt 5041 gcgggcaggg aagaaaatcg cttaccaggg ccgtgtcaag gacgagccag cctactactg 5101 caacgagtgc gatgtggagg tgtttaacat cctgttcgtg acaagtgaga atggcagccg 5161 caacacgtac ctggtacact gcgagggctg tgcccggcgc cgcagcgcag gcctgcaggg 5221 cgtggtggtg ctggagcagt accgcactga ggagctggct caggcctacg acgccttcac 5281 gctggtgagg gcccggcggg cgcgcgggca gcggaggagg gcactggggc aggctgcagg 5341 gacgggcttc gggagcccgg ccgcgccttt ccctgagccc ccgccggctt tctcccccca 5401 ggccccagcc agcacgtcgc gatgaggccg gacgccccgc ccgcctgcct gcccgcgcaa 5461 ggcgccgcgg ggccaccagc acatgcctgg gctggaccta ggtcccgcct gtggccgaga 5521 agggggtcgg gcccagccct tccaccccat tggcagctcc cctcacttaa tttattaaga 5581 aaaacttttt tttttttttt agcaaatatg aggaaaaaag gaaaaaaaat gggagacggg 5641 ggagggggct ggcagcccct cgcccaccag cgcctcccct caccgacttt ggccttttta 5701 gcaacagaca caaggaccag gctccggcgg cggcgggggt cacatacggg ttccctcacc 5761 ctgccagccg cccgcccgcc cggcgcagat gcacgcggct cgtgtatgta catagacgtt 5821 acggcagccg aggtttttaa tgagattctt tctatgggct ttacccctcc cccggaacct 5881 ccttttttac ttccaatgct agctgtgacc cctgtacatg tctctttatt cacttggtta 5941 tgatttgtat tttttgttct tttcttgttt ttttgttttt aatttataac agtcccactc 6001 acctctattt attcattttt gggaaaaccc gacctcccac acccccaagc catcctgccc 6061 gcccctccag ggaccgcccg tcgccgggct ctccccgcgc cccagtgtgt gtccgggccc 6121 ggcccgaccg tctccacccg tccgcccgcg gctccagccg ggttctcatg gtgctcaaac 6181 ccgctcccct cccctacgtc ctgcactttc tcggaccagt ccccccactc ccgacccgac 6241 cccagcccca cctgagggtg agcaactcct gtactgtagg ggaagaagtg ggaactgaaa 6301 tggtattttg taaaaaaaat aaataaaata aaaaaattaa aggttttaaa gaaagaacta 6361 tgaggaaaag gaaccccgtc cttcccagcc ccggccaact ttaaaaaaca cagaccttca 6421 cccccacccc cttttctttt taagtgtgaa acaacccagg gccagggcct cactggggca 6481 gggacacccc ggggtgagtt tctctggggc tttattttcg ttttgttggt tgttttttct 6541 ccacgctggg gctgcggagg ggtggggggt ttacagtccc gcaccctcgc actgcactgt 6601 ctctctgccc caggggcaga ggggtcttcc caaccctacc cctattttcg gtgatttttg 6661 tgtgagaata ttaatattaa aaataaacgg agaaaaaaaa aaaaaaaaaa aaaaaaaaaa 6721 aaaaaaaaaa a

By “KDM6C polypeptide” (histone demethylase UTY, also referred to as ubiquitously-transcribed TPR protein on the Y chromosome) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: O14607.2, or a fragment thereof, and having demethylase activity. An exemplary KDM6C amino acid sequence is provided below (SEQ ID NO: 8):

1 mkscavsltt aavafgdeak kmaegkasre seeesvsltv eerealggmd srlfgfvrlh 61 edgartktll gkavrcyesl ilkaegkves dffcqlghfn llledyskal sayqryyslq 121 adywknaafl yglglvyfyy nafhwaikaf qdvlyvdpsf crakeihlrl glmfkvntdy 181 ksslkhfqla lidcnpctls naeiqfhiah lyetqrkyhs akeayeqllq tenlpaqvka 241 tvlqqlgwmh hnmdlvgdka tkesyaiqyl qksleadpns gqswyflgrc yssigkvqda 301 fisyrqsidk seasadtwcs igvlyqqqnq pmdalqayic avqldhghaa awmdlgtlye 361 scnqpqdaik cylnaarskr csntstlaar ikflqngsdn wnggqslshh pvqqvyslcl 421 tpqklqhleq lranrdnlnp aqkhqleqle sqfvlmqqmr hkevaqvrtt gihngaitds 481 slptnsvsnr qphgaltrvs svsqpgvrpa cvekllssga fsagcipcgt skilgstdti 541 llgsnciags esngnvpylq qnthtlphnh tdlnssteep wrkqlsnsaq glhksqsscl 601 sgpneeqplf stgsaqyhqa tstgikkane hltlpsnsvp qgdadshlsc htatsggqqg 661 imftkeskps knrslvpets rhtgdtsngc advkglsnhv hqliadavss pnhgdspnll 721 iadnpqlsal ligkangnvg tgtcdkvnni hpavhtktdh svasspssai statpspkst 781 eqrsinsvts lnsphsglht vngeglgksq sstkvdlpla shrstsqilp smsvsicpss 841 tevlkacrnp gknglsnsci lldkcppprp ptspypplpk dklnpptpsi ylenkrdaff 901 pplhqfctnp knpvtvirgl agalkldlgl fstktlvean nehmvevrtq llqpadenwd 961 ptgtkkiwrc esnrshttia kyaqyqassf qeslreenek rtqhkdhsdn estssensgr 1021 rrkgpfktik fgtnidlsdn kkwklqlhel tklpafarvv sagnllthvg htilgmntvq 1081 lymkvpgsrt pghqennnfc svninigpgd cewfvvpedy wgvlndfcek nnlnflmssw 1141 wpnledlyea nvpvyrfiqr pgdlvwinag tvhwvqavgw cnniawnvgp ltacqyklav 1201 eryewnklks vkspvpmvhl swnmarnikv sdpklfemik ycllkilkqy qtlrealvaa 1261 gkeviwhgrt ndepahycsi cevevinllf vtnesntqkt yivhchdcar ktskslenfv 1321 vleqykmedl iqvydqftla lslssss

By “KDM6C polynucleotide is meant a nucleic acid molecule encoding a KDM6C polypeptide. An exemplary KDM6A polynucleotide sequence is provided at NM_001258249.1, which sequence is reproduced below (SEQ ID NO: 9):

1 gctcatcgtt tgttgtttag ataatatcat gaactgataa atgcagttgc cacgttgatt 61 ccctagggcc tggcttaccg actgaggtca taagatatta tgccttctct ttagacttgg 121 tcagtggaga ggaaatgggc aaagaaccag cctatggagg tgacaaggcc ttagggccaa 181 aagtcttgag ggtgaaggtt tagggcctgc gcagcttccc tgccatgccc cgcaaggtct 241 cgcattcgca aggcttgtga cagtgggagc ctcattacgg actctcctaa agtccatggt 301 gtcctctttt cgcatttgcg ccccgtgggt gatgcccgat gccgcccttc ccatcgctct 361 cttccccttc aagcgtatcg caactgcaaa aacacccagc acagacactc cattttctat 421 cttaatgcat ttaactagca caacctacag gttgttccat cccagagact acccttttct 481 ccatagacgt gaccatcaac caaccagcgg tcagaatcag tcagcctctg tcatgttcct 541 aggtccttgg cgaactggct gggcggggtc ccagcagcct aggagtacag tggagcaatg 601 cctgacgtaa gtcaacaaag atcacgtgag acgaatcagt cgcctagatt ggctacaact 661 aagtggttgg gagcggggag gtcgcggcgg ctgcgtgggg ttcgcccgtg acacaattac 721 aactttgtgc tggtgctggc aaagtttgtg attttaagaa attctgctgt gctctccagc 781 actgcgagct tctgccttcc ctgtagtttc ccagatgtga tccaggtagc cgagttccgc 841 tgcccgtgct tcggtagctt aagtctttgc ctcagctttt ttccttgcag ccgctgagga 901 ggcgataaaa ttggcgtcac agtctcaagc agcgattgaa ggcgtctttt caactactcg 961 attaaggttg ggtatcgtcg tgggacttgg aaatttgttg tttccatgaa atcctgcgca 1021 gtgtcgctca ctaccgccgc tgttgccttc ggtgatgagg caaagaaaat ggcggaagga 1081 aaagcgagcc gcgagagtga agaggagtct gttagcctga cagtcgagga aagggaggcg 1141 cttggtggca tggacagccg tctcttcggg ttcgtgaggc ttcatgaaga tggcgccaga 1201 acgaagaccc tactaggcaa ggctgttcgc tgctacgaat ctttaatctt aaaagctgaa 1261 ggaaaagtgg agtctgactt cttttgccaa ttaggtcact tcaacctctt gttggaagat 1321 tattcaaaag cattatctgc atatcagaga tattacagtt tacaggctga ctactggaag 1381 aatgctgcgt ttttatatgg ccttggtttg gtctacttct actacaatgc atttcattgg 1441 gcaattaaag catttcaaga tgtcctttat gttgacccca gcttttgtcg agccaaggaa 1501 attcatttac gacttgggct catgttcaaa gtgaacacag actacaagtc tagtttaaag 1561 cattttcagt tagccttgat tgactgtaat ccatgtactt tgtccaatgc tgaaattcaa 1621 tttcatattg cccatttgta tgaaacccag aggaagtatc attctgcaaa ggaggcatat 1681 gaacaacttt tgcagacaga aaaccttcct gcacaagtaa aagcaactgt attgcaacag 1741 ttaggttgga tgcatcataa tatggatcta gtaggagaca aagccacaaa ggaaagctat 1801 gctattcagt atctccaaaa gtctttggag gcagatccta attctggcca atcgtggtat 1861 tttcttggaa ggtgttattc aagtattggg aaagttcagg atgcctttat atcttacagg 1921 caatctattg ataaatcaga agcaagtgca gatacatggt gttcaatagg tgtgttgtat 1981 cagcagcaaa atcagcctat ggatgcttta caggcatata tttgtgctgt acaattggac 2041 catgggcatg ccgcagcctg gatggaccta ggtactctct atgaatcctg caatcaacct 2101 caagatgcca ttaaatgcta cctaaatgca gctagaagca aacgttgtag taatacctct 2161 acgcttgctg caagaattaa atttctacag gctcagttgt gtaaccttcc acaaagtagt 2221 ctacagaata aaactaaatt acttcctagt attgaggagg catggagcct accaatcccc 2281 gcagagctta cctccaggca gggtgccatg aacacagcac agcaggctta tagagctcat 2341 gatccaaata ctgaacatgt attaaaccac agtcaaacac caattttaca gcaatccttg 2401 tcactacaca tgattacttc tagccaagta gaaggcctgt ccagtcctgc caagaagaaa 2461 agaacatcta gtccaacaaa gaatggttct gataactgga atggtggcca gagtctttca 2521 catcatccag tacagcaagt ttattcgttg tgtttgacac cacagaaatt acagcacttg 2581 gaacaactgc gagcaaatag agataattta aatccagcac agaagcatca gctggaacag 2641 ttagaaagtc agtttgtctt aatgcagcaa atgagacaca aagaagttgc tcaggtacga 2701 actactggaa ttcataacgg ggccataact gattcatcac tgcctacaaa ctctgtctct 2761 aatcgacaac cacatggtgc tctgaccaga gtatctagcg tctctcagcc tggagttcgc 2821 cctgcttgtg ttgaaaaact tttgtccagt ggagcttttt ctgcaggctg tattccttgt 2881 ggcacatcaa aaattctagg aagtacagac actatcttgc taggcagtaa ttgtatagca 2941 ggaagtgaaa gtaatggaaa tgtgccttac ctgcagcaaa atacacacac tctacctcat 3001 aatcatacag acctgaacag cagcacagaa gagccatgga gaaaacagct atctaactcc 3061 gctcaggggc ttcataaaag tcagagttca tgtttgtcag gacctaatga agaacaacct 3121 ctgttttcca ctgggtcagc ccagtatcac caggcaacta gcactggtat taagaaggcg 3181 aatgaacatc tcactctgcc tagtaattca gtaccacagg gggatgctga cagtcacctc 3241 tcctgtcata ctgctacctc aggtggacaa caaggcatta tgtttaccaa agagagcaag 3301 ccttcaaaaa atagatcctt ggtgcctgaa acaagcaggc atactggaga cacatctaat 3361 ggctgtgctg atgtcaaggg actttctaat catgttcatc agttgatagc agatgctgtt 3421 tccagtccta accatggaga ttcaccaaat ttattaattg cagacaatcc tcagctctct 3481 gctttgttga ttggaaaagc caatggcaat gtgggtactg gaacctgtga caaagtgaat 3541 aatattcacc cagctgttca tacaaagact gatcattctg ttgcctcttc accctcttca 3601 gccatttcca cagcaacacc ttctcctaaa tccactgagc agagaagcat aaacagtgtt 3661 accagcctta acagtcctca cagtggatta cacacagtca atggagaggg gctggggaag 3721 tcacagagct ctacaaaagt agacctgcct ttagctagcc acagatctac ttctcagatc 3781 ttaccatcaa tgtcagtgtc tatatgcccc agttcaacag aagttctgaa agcatgcagg 3841 aatccaggta aaaatggctt gtctaatagc tgcattttgt tagataaatg tccacctcca 3901 agaccaccaa cttcaccata cccacccttg ccaaaggaca agttgaatcc acccacacct 3961 agtatttact tggaaaataa acgtgatgct ttctttcctc cattacatca attttgtaca 4021 aatccaaaaa accctgttac agtaatacgt ggccttgctg gagctcttaa attagatctt 4081 ggacttttct ctaccaaaac tttggtagaa gctaacaatg aacatatggt agaagtgagg 4141 acacagttgc tgcaaccagc agatgaaaac tgggatccca ctggaacaaa gaaaatctgg 4201 cgttgtgaaa gcaatagatc tcatactaca attgccaaat acgcacaata ccaggcttcc 4261 tccttccagg aatcattgag agaagaaaat gagaaaagaa cacaacacaa agatcattca 4321 gataacgaat ccacatcttc agagaattct ggaaggagaa ggaaaggacc ttttaaaacc 4381 ataaaatttg ggaccaacat tgacctctct gataacaaaa agtggaagtt gcagttacat 4441 gaactgacta aacttcctgc ttttgcgcgt gtggtgtcag caggaaatct tctaacccat 4501 gttgggcata ccattctggg catgaataca gtacaactgt atatgaaagt tccagggagt 4561 cggacaccag gtcaccaaga aaataacaac ttctgctctg ttaacataaa tattggtcca 4621 ggagattgtg aatggtttgt tgtacctgaa gattattggg gtgttctgaa tgacttctgt 4681 gaaaaaaata atttgaattt tttaatgagt tcttggtggc ccaaccttga agatctttat 4741 gaagcaaatg tccctgtgta tagatttatt cagcgacctg gagatttggt ctggataaat 4801 gcaggcactg tgcattgggt tcaagctgtt ggctggtgca ataacattgc ctggaatgtt 4861 ggtccactta cagcctgcca gtataaattg gcagtggaac ggtatgaatg gaacaaattg 4921 aaaagtgtga agtcaccagt acccatggtg catctttcct ggaatatggc acgaaatatc 4981 aaagtctcag atccaaagct ttttgaaatg attaagtatt gtcttttgaa aattctgaag 5041 caatatcaga cattgagaga agctcttgtt gcagcaggaa aagaggttat atggcatggg 5101 cggacaaatg atgaaccagc tcattactgt agcatttgtg aggtggaggt ttttaatctg 5161 ctttttgtca ctaatgaaag caatactcaa aaaacctaca tagtacattg ccatgattgt 5221 gcacgaaaaa caagcaaaag tttggaaaat tttgtggtgc tcgaacagta caaaatggag 5281 gacctaatcc aagtttatga tcaatttaca ctagctcttt cattatcatc ctcatcttga 5341 tatagttcca tgaatattaa atgagattat ttctgctctt caggaaattt ctgcaccact 5401 ggttttgtag ctgtttcata aaactgttga ctaaaagcta tgtctatgca accttccaag 5461 aatagtatgt caagcaactg gacacagtgc tgcctctgct tcaggactta acatgctgat 5521 ccagctgtac ttcagaaaaa taatattaat catatgtttt gtgtacgtat gacaaactgt 5581 caaagtgaca cagaatactg atttgaagat agcctttttt atgtttctct atttctgggc 5641 tgatgaatta atattcattt gtattttaac cctgcagaat tttccttagt taaaaacact 5701 ttcctagctg gtcatttctt cataagatag caaatttaaa tctctcctcg atcagctttt 5761 aaaaaatgtg tactattatc tgaggaagtt ttttactgct ttatgttttt gtgtgttttg 5821 aggccatgat gattacattt gtggttccaa aataattttt ttaaatatta atagcccata 5881 tacaaagata atggattgca catagacaaa gaaataaact tcagatttgt gatttttgtt 5941 tctaaacttg atacagattt acactattta taaatacgta tttattgcct gaaaatattt 6001 gtgaatggaa tgttgttttt ttccagacgt aactgccatt aaatactaag gagttctgta 6061 gttttaaaca ctactcctat tacattttat atgtgtagat aaaactgctt agtattatac 6121 agaaattttt attaaaattg ttaaatgttt aaagggtttc ccaatgtttg agtttaaaaa 6181 agactttctg aaaaaatcca ctttttgttc attttcaaac ctaatgatta tatgtatttt 6241 atatgtgtgt gtatgtgtac acacatgtat aatatataca gaaacctcga tatataattg 6301 tatagatttt aaaagtttta ttttttacat ctatggtagt ttttgaggtg cctattataa 6361 agtattacgg aagtttgctg tttttaaagt aaatgtcttt tagtgtgatt tattaagttg 6421 tagtcaccat agtgatagcc cataaataat tgctggaaaa ttgtatttta taacagtaga 6481 aaacatatag tcagtgaagt aaatatttta aaggaaacat tatatagatt tgataaatgt 6541 tgtttataat taagagtttc ttatggaaaa gagattcaga atgataacct cttttagaga 6601 acaaataagt gacttatttt tttaaagcta gatgactttg aaatgctata ctgtcctgct 6661 tgtacaacat ggtttggggt gaaggggagg aaagtattaa aaaatctata tcgctagtaa 6721 attgtaataa gttctattaa aacttgtatt tcatatgaaa aatttgctaa tttaatatta 6781 actcatttga taataatact tgtcttttct acctctc

By “Gab1 polypeptide” (GRB2-associated-binding protein 1) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP_997006.1, or a fragment thereof. An exemplary Gab1 amino acid sequence is provided below (SEQ ID NO: 10):

1 msggevvcsg wlrksppekk lkryawkrrw fvlrsgrltg dpdvleyykn dhakkpirii 61 dlnlcqqvda gltinkkefe nsyifdinti drifylvads eeemnkwvrc icdicgfnpt 121 eedpvkppgs slqapadlpl aintappstq adsssatlpp pyqlinvpph letlgiqedp 181 qdylllincq skkpeptrth adsakstsse tdcndnvpsh knpassqskh gmngffqqqm 241 iydsppsrap sasvdsslyn lprsyshdvl pkvspsstea dgelyvfntp sgtssvetqm 301 rhvsisydip ptpgntyqip rtfpegtlgq tskldtipdi ppprppkphp ahdrspvetc 361 siprtasdtd ssyciptagm spsrsntist vdlnklrkda ssqdcydipr afpsdrsssl 421 egfhnhfkvk nvltvgsvss eeldenyvpm npnspprqhs ssftepigea nyvpmtpgtf 481 dfssfgmqvp ppahmgfrss pktpprrpvp vadcepppvd rnlkpdrkgq spkilrlkph 541 glertdsqti gdfatrrkvk papleikplp eweelqapvr spitrsfard ssrfpmsprp 601 dsvhsttsss dshdseenyv pmnpnlssed pnlfgsnsld ggsspmikpk gdkqveyldl 661 dldsgkstpp rkqkssgsgs svadervdyv vvdqqktlal kstreawtdg rqstesetpa 721 ksvk

By “Gab1 polynucleotide” is meant a nucleic acid molecule encoding a Gab1 polypeptide. An exemplary Gab1 polynucleotide sequence is provided at NM_002039.3, which is reproduced below (SEQ ID NO: 11):

1 agggggcgga gcgcaaagga cagaagctcc ggcaccgagt cggggcagag tcccgctgag 61 tccgagcgct gctgaggcag ctggcgagac ggcacgtctg gaggcgaggc gggcgcactg 121 aaaggaggcc ggcgcgcccg cggccccggc tcgcgttctg ttcaggttcg tgggcctgca 181 gaggagagac tcgaactcgt ggaacccgcg caccgtggag tctgtccgcc cagtccgtcc 241 ggggtgcgcg accaggagag ctaggttctc gccactgcgc gctcggcagg cgtcggctgt 301 gtcgggagcg cgcccgccgc ccctcagctg cccggcccgg agcccgagac gcgcgcacca 361 tgagcggtgg tgaagtggtc tgctccggat ggctccgcaa gtcccccccg gagaaaaagt 421 tgaagcgtta tgcatggaag aggagatggt tcgtgttacg cagtggccgt ttaactggag 481 atccagatgt tttggaatat tacaaaaatg atcatgccaa gaagcctatt cgtattattg 541 atttaaattt atgtcaacaa gtagatgctg gattgacatt taacaaaaaa gagtttgaaa 601 acagctacat ttttgatatc aacactattg accggatttt ctacttggta gcagacagcg 661 aggaggagat gaataagtgg gttcgttgta tttgtgacat ctgtgggttt aatccaacag 721 aagaagatcc tgtgaagcca cctggcagct ctttacaagc accagctgat ttacctttag 781 ctataaatac agcaccacca tccacccagg cagattcatc ctctgctact ctacctcctc 841 catatcagct aatcaatgtt ccaccacacc tggaaactct tggcattcag gaggatcctc 901 aagactacct gttgctcatc aactgtcaaa gcaagaagcc cgaacccacc agaacgcatg 961 ctgattctgc aaaatccacc tcttctgaaa cagactgcaa tgataacgtc ccttctcata 1021 aaaatcctgc ttcctcccag agcaaacatg gaatgaatgg cttttttcag cagcaaatga 1081 tatacgactc tccaccttca cgtgccccat ctgcttcagt tgactccagc ctttataacc 1141 tgcccaggag ttattcccat gatgttttac caaaggtgtc tccatcaagt actgaagcag 1201 atggagaact ctatgttttt aataccccat ctgggacatc gagtgtagag actcaaatga 1261 ggcatgtatc tattagttat gacattcctc caacacctgg taatacttat cagattccac 1321 gaacatttcc agaaggaacc ttgggacaga catcaaagct agacactatt ccagatattc 1381 ctccacctcg gccaccgaaa ccacatccag ctcatgaccg atctcctgtg gaaacgtgta 1441 gtatcccacg caccgcctca gacactgaca gtagttactg tatccctaca gcagggatgt 1501 cgccttcacg tagtaatacc atttccactg tggatttaaa caaattgcga aaagatgcta 1561 gttctcaaga ctgctatgat attccacgag catttccaag tgatagatct agttcacttg 1621 aaggcttcca taaccacttt aaagtcaaaa atgtgttgac agtgggaagt gtttcaagtg 1681 aagaactgga tgaaaattac gtcccaatga atcccaattc accaccacga caacattcca 1741 gcagttttac agaaccaatt caggaagcaa attatgtgcc aatgactcca ggaacatttg 1801 atttttcctc atttggaatg caagttcctc ctcctgctca tatgggcttc aggtccagcc 1861 caaaaacccc tcccagaagg ccagttcctg ttgcagactg tgaaccaccc cccgtggata 1921 ggaacctcaa gccagacaga aaagtcaagc cagcgccttt agaaataaaa cctttgccag 1981 aatgggaaga attacaagcc ccagttagat ctcccatcac taggagtttt gctcgagact 2041 cttccaggtt tcccatgtcc ccccgaccag attcagtgca tagcacaact tcaagcagtg 2101 actcacacga cagtgaagag aattatgttc ccatgaaccc aaacctgtcc agtgaagacc 2161 caaatctctt tggcagtaac agtcttgatg gaggaagcag ccctatgatc aagcccaaag 2221 gagacaaaca ggtggaatac ttagatctcg acttagattc tgggaaatcc acaccaccac 2281 gtaagcaaaa gagcagtggc tcaggcagca gtgtagcaga tgagagagtg gattatgttg 2341 ttgttgacca acagaagacc ttggctctaa agagtacccg ggaagcctgg acagatggga 2401 gacagtccac agaatcagaa acgccagcga agagtgtgaa atgaaaatat tgccttgcca 2461 tttctgaaca aaagaaaact gaattgtaaa gataaatccc ttttgaagaa tgacttgaca 2521 cttccactct aggtagatcc tcaaatgagt agagttgaag tcaaaggacc tttctgacat 2581 aatcaagcaa tttagactta agtggtgctt tgtggtatct gaacaattca taacatgtaa 2641 ataatgtggg aaaatagtat tgtttagctc ccagagaaac atttgttcca cagttaacac 2701 actcgtagta ttactgtatt tatgcacttt ttcatctaaa acattgttct gggttttccc 2761 aatgtacctt accataattc ctttgggagt tcttgttttt tgtcacacta ctttatataa 2821 caatactaag tcaactaagc tacttttaga tttggaaatt gctgtttaca gtctaacaac 2881 attaaaatga gaggtagatt cacaagttag ctttctacct gaagcttcag gtgataacca 2941 ttagcttata cttggactca tcatttgttg ccttccaaaa tgctgaggat aatgtatgta 3001 ctggtgtcag gacctagttc tctggttaat gtacatttag tttttaatgg tggaactttg 3061 ttatattttg ttaattacag tgtttttggt tcattgagtg aagattctgc cgggtgggat 3121 cttgcacctt tgaaagactg aataattaca ctaccaagta agcctgcaaa tcattgatgg 3181 catgcagtga tgatgtgctc ttacacttgt taacatgtat taagtgttat ttgcaaaagg 3241 tagattatgt aaccaatcag gtacgtacca ggcagtgatg tgctaataca ctgatcaggt 3301 ttagacaatg agctttggtt gtgttcttgt tagtcctaat attggttttc agtttggaat 3361 taataaagca gttgacattc actgttagtt acagcaacat actgtgattt ttaattagat 3421 agtaattcag atttattact ctatgaaatt ctgtcttttg acaccatagt gccctttcta 3481 tgattttttt tacttaatat tcttcttggc cttatattta attccctatg caattaatat 3541 tttatatctg cattttttta aaaaaaatag atgttatata agtgattctc gtatgtagca 3601 cctgttgctt ttccactgaa agaattacgg attttgtact gtgatttata ttcactgccc 3661 caattcaaga aatattggag ccttgctaca atgtgaaatg ttatagtcat ggactccttc 3721 caaccagatt tctgaaaaca ccagagggat ggtataattc tgtctcacct ataacatggt 3781 cctgtgacat agatattaag accacaagtt gtagtgaggc tacaattata ttcgtctgtc 3841 ttggctttgc aacataattt agaaagcacg tatagttgtt ttttaaccaa gttacataca 3901 atctcatgta ctgatttgag acttataaca atttttggag ggggcataga gaaaggagtg 3961 cccacagttg aggcatgacc ccctccattc agacctctaa ctgttgcctg agtacacaga 4021 tgtgccctga tttctggccc attggccata gtactgtgcc taatcaatgt aataggttta 4081 ttttcccaat cctcaaacta aaaatgttca taacaagatg aattgtagac tagtaacatt 4141 tgatgctttt aaatatttgc ttctttttaa acaaaaacta aaacccagaa gtgaattttt 4201 aggtggattt ttaaataaaa aagattgatt gagtttggtg tgcaagctgt tttataatga 4261 aacaacaaaa tgaaatctaa aatcctgaaa tgtgcctaaa ctatcaaaac acacgataca 4321 gctaatgtgt aaagatgcta aattctgtta cttggaggat gaatatattt aagatttaaa 4381 acacaataat aaatacatga ttaattcaaa aataaaaatc tttacagctg cctatcaagg 4441 gtctaaagca cttaatgaat gtttttagtc taacttatca ttaacttttt acaagtcacc 4501 atatttgaag atctgtagca ctctgatttt cagaaaattt ttcattctga ataatttaaa 4561 aatggtgatg tattagaaag gcagtttgct ttagaaaact aaatcacatt gaacattgta 4621 ttagagaatt aaattaaaag tttcttacag agcagtattt tccaaacatt tttagcacta 4681 gaatcttttt agatgaaatt ttatgtataa ccccaataca taaagcctga aaactcaatt 4741 ttatcaatat aaatgtattt tgggttcaca tttatgctta ttcattttgg ctcattacta 4801 agcataataa gattctgagt tatttctgaa taacacaaat gtggagttat acatagttga 4861 tgaaaccagc agccaattta tagctatgcc ctgttttatt tgtatactat caagaaaatt 4921 ttgattcaca caaatgtaag caaaaataat aggttttaaa catacatctc aggaaattct 4981 ttaattagag atagctaaag ttattcaagg tctatacaaa aataagttat cctggtagtg 5041 gaagttaata cataagcagt ctccagtgtg gtaaagtagg gtatgtaaca catcagaatg 5101 tgcgttttta ttaggtttta aaatatgcac gtataaaaac taaatttgaa tcaaaccctt 5161 ttaactcacc tccaagaagc tagactttgg ccaggaatgg gctaaaaacc actggttaac 5221 gatgtgacag ttatgatctt ggagattgga aatctttctt ccacattaga gttctttacc 5281 ttaattcctt attctgaaaa attgtaagat tttatgaagg tttgaatact gaagcacagt 5341 tctgctttca aaaattaaaa ttcaaacttg aaaaagctgt ttaacccatg gaagatatca 5401 tttagtaaga tgtaaaagat tttttaaatc tacacttcag tttatacatc tttatcatta 5461 tcaatactat ataagttact gtgagcattt tagagaattc cataaaggta ctatgagtgt 5521 gtctgtatgt gtgtgtatat atagcattgt atttaatcat agactaaatt taatttgata 5581 tagaaatact actttacttg tacattaagg tcataatttc tgctggactc ttttatattt 5641 aattaatggg gattatagtc ttccttcata aatgcattta aacctgaaat tgaacaccag 5701 tgtttttctt tttctactta tgggaagttg tctgcttccc cctttagaga aaacagtatt 5761 tttatatttt gttaaaatat taactacttt atgcctacac actatgctgt agatactgat 5821 cataattctt gggtgttcac aaacactcct agtgcctctt ttttggcccg ttgaaagtgt 5881 tggtattact actttcacta cagagccttt ggccctctaa taatgctgag gtgggctgat 5941 ccttcccatt tctgtcttcg ggtcattctg gtaggtcttc tcctccactg tcaagtaagc 6001 aatcaggtcc gtgacaggga ttggacatat gaacaaatta agtggataca cacagtgaga 6061 aagatacatg cattctatgg taacaactac tgtcaataac atctgatgtt acatgcacat 6121 ttatatatat ataattttaa aaactgaact atgagaagcc atggtataaa tgaatattgt 6181 ggacatcatg gacttgatat gatagaaatc aattgtcagc ttgagaaagt tgtttttaat 6241 ctgtctaaat agttcatgca ttactacagt taaaaatagt ttcatttgtc ttctatagac 6301 ttaattttat tccggttcag tataatctct gttaacagag tttcagcaaa ctgattggtc 6361 aaggtattaa catagcttct acttccttta cttaaaaaga tgtggtttta tgtaagttct 6421 tgattactga tgatcatccc aaattttgac aacaaaatca tatgtataaa tttatttctc 6481 ccctcttgtt catcatcttt tgtaaaggtc ccattgtaga tcttttctgc taccaaataa 6541 aacttttcaa acaatttggt ttcaagacct taaatagaca agttggatac taagattgtg 6601 aactgataag gacatataaa tttatatttc cagcccttcc ttagagtctt tatctgcatc 6661 aaaaacccaa ttctgccatt aactgtgctt cccagtccca cctctatatg tcactcattt 6721 tctgcaacaa agatctcact aaatcatgtt gaaacacaag tcatgatcct ctctaagtaa 6781 atagaaaaag ctccctggaa aaactctgtt gccacatgca cgtgccctgt tactcctcca 6841 gccagccagt gctgccagca ttttattgtg taaaagtcca aataaataag ggcctgcatg 6901 caacctttat cttcagaaac taggttttat atgtaaaatg tgacttggga aatgattctg 6961 tttattaact ggctgggatt tttcatttct atgaaagttt caaacatctc cagtacttta 7021 taaaatccca acaattgctg taagtcagca ctttggtcca ctcagcccac ccagcccact 7081 tgcaactctg actcttcact gaatcatatt tgggaagttt gggtagggtg aggctatctt 7141 cttcaagatt attttctcat atgtctgtct gtcaccttgt aaaccatgag actcctgggt 7201 atttgcatgt aacttctttg aggaagttac caccatctct gatatagaca cactttttga 7261 gttgcagttt ctgttagaat tttttggaga ctaacttgcc aattctgtga atgttattga 7321 atatttaaaa agctgggtct gtaatgggag gcattttatt agctgttgtg attgggtaac 7381 atgtcccctt agatttcctg atttaaaatt atacaaaatt actatttttg ataaaataaa 7441 ggaacaccta cagaaaatta agtttctaag atgtttctat acttcattag aaaagatttt 7501 attactatta cttatggtta ttggtgatta acacttaatg cgtctcctct gattttgtgt 7561 tccatgaggt gcttggaaca tttggagtgc tctgtgcgag ggacatacag tgatatagga 7621 aatttaaaaa ttaaaataat acccaaaacc cactttatca gatatggtat tgtgatggtt 7681 aatattatgt gtcaacttgg tgaggctatg gcgcccatgt gtttggtcaa acactagcct 7741 agatgttgct gtgaatatat tttgtagatg tgattaacat ttacaatcag ttgattttaa 7801 gtaaagcaga ttctcatcca aaaaaaaaaaaaaaaa

By “Sfmbt2 polypeptide” (scm-like with four MBT domains protein 2) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP_001018049.1, or a fragment thereof. An exemplary Sfmbt2 amino acid sequence is provided below (SEQ ID NO: 12):

1 mestlsasnm qdpsssplek clgsangngd ldseegssle etgfnwgeyl eetgasaaph 61 tsfkhveisi qsnfqpgmkl evanknnpdt ywvatiittc gqllllrycg ygedrradfw 121 cdvviadlhp vgwctqnnkv lmppdaikek ytdwteflir dltgsrtapa nllegplrgk 181 gpidlitvgs lielqdsqnp fqywivsvie nvggrlrlry vgledtesyd qwlfyldyrl 241 rpvgwcqenk yrmdppseiy plkmasewkc tlekslidaa kfplpmevfk dhadlrshff 301 tvgmkletvn mcepfyispa svtkvinnhf fqvtiddlrp epsklsmlch adslgilpvq 361 wclkngvslt ppkgysgqdf dwadyhkqhg aqeappfcfr ntsfsrgftk nmkleavnpr 421 npgelcvasv vsvkgrlmwl hleglqtpvp evivdvesmd ifpvgwcean sypltaphkt 481 vsqkkrkiav vqpekqlppt vpvkkiphdl clfphldttg tvngkyccpq lfinhrcfsg 541 pylnkgriae lpqsvgpgkc vlvlkevlsm iinaaykpgr vlrelqlved phwnfqeetl 601 kakyrgktyr avvkivrtsd qvanfcrrvc akleccpnlf spvlisencp encsihtktk 661 ytyyygkrkk iskppigesn pdsghpkpar rrkrrksifv qkkrrssavd ftagsgeese 721 eedadamddd taseetgsel rddqtdtssa evpsarprra vtlrsgsepv rrpppertrr 781 grgapaassa eegekcpptk pegtedtkqe eeerlvlesn plewtvtdvv rfikltdcap 841 lakifqeqdi dgqalllltl ptvqecmelk lgpaiklchq iervkvafya qyan

By “Sfmbt2 polynucleotide” is meant a polypeptide encoding an Sfmbt2 polypeptide. An exemplary Sfmbt2 polynucleotide sequence is provided at NM_001018039.1, which is reproduced below (SEQ ID NO: 13):

1 cgccttgtgt gtgctggatc ctgcgcgggt agatccccga gtaatttttt ctgcaggatg 61 aattaagaga agagacactt gctcatcagg catggagagc actttgtcag cttccaatat 121 gcaagaccct tcatcttcac ccttggaaaa gtgtctcggc tcagctaatg gaaatggaga 181 ccttgattct gaagaaggct caagcttgga ggaaactggc tttaactggg gagaatattt 241 ggaagagaca ggagcaagtg ctgctcccca cacatcattc aaacacgttg aaatcagcat 301 tcagagcaac ttccagccag gaatgaaatt ggaagtggct aataagaaca acccggacac 361 gtactgggtg gccacgatca ttaccacgtg cgggcagctg ctgcttctgc gctactgcgg 421 ttacggggag gaccgcaggg ccgacttctg gtgtgacgta gtcatcgcgg atttgcaccc 481 cgtggggtgg tgcacacaga acaacaaggt gttgatgccg ccggacgcaa tcaaagagaa 541 gtacacagac tggacagaat ttctcatacg tgacttgact ggttcgagga cagcacccgc 601 caacctcctg gaaggtcctc tgcgagggaa aggccctata gacctcatta cagttggttc 661 cttaatagaa cttcaggatt cccagaaccc ttttcagtac tggatagtta gtgtgattga 721 aaatgttgga ggaagattac gccttcgcta tgtgggattg gaggacactg aatcctatga 781 ccagtggttg ttttacttgg attacagact tcgaccagtt ggttggtgtc aagagaataa 841 atacagaatg gacccacctt cagaaatcta tcctttgaag atggcctctg aatggaaatg 901 tactctggaa aaatccctta ttgatgctgc caaatttcct cttccaatgg aagtgtttaa 961 ggatcacgca gatttgcgaa gccatttctt cacagttggg atgaagcttg agacagtgaa 1021 tatgtgcgag cccttttaca tctctcctgc gtcggtgact aaggttttta acaatcactt 1081 ttttcaagtg actattgatg acctaagacc tgaaccaagt aaactgtcaa tgctgtgcca 1141 tgcagattct ttggggattt tgccagtaca gtggtgcctt aaaaatggag tcagcctcac 1201 tcctcccaaa ggttactctg gccaggactt cgactgggca gattatcaca agcagcatgg 1261 ggcgcaggaa gcccctccct tctgcttccg aaatacatca ttcagtcgag gtttcacaaa 1321 gaacatgaaa cttgaagctg tgaaccccag gaatccagga gaactgtgtg tggcctccgt 1381 tgtgagtgtg aaggggcggc taatgtggct tcacctggaa gggctgcaga ctcctgttcc 1441 agaggtcatt gttgatgtgg aatccatgga catcttccca gtgggctggt gtgaagccaa 1501 ttcttatcct ttgactgcac cacacaaaac agtctcacaa aagaagagaa agattgcagt 1561 cgtgcaacca gagaaacaat tgccgcccac agtgcctgtt aagaaaatac ctcatgacct 1621 ttgtttattc cctcacctgg acaccacagg aaccgtcaac gggaaatact gctgtcctca 1681 gctcttcatc aaccacaggt gtttctcagg cccttacctg aacaaaggaa ggattgcaga 1741 gctacctcag tcggtgggac cgggcaaatg cgtgctggtt cttaaagagg ttcttagcat 1801 gataatcaac gcagcctaca agcctggaag ggtattaaga gaattacagc tggtagaaga 1861 tccccactgg aatttccagg aagagacgct gaaggccaaa tacagaggca aaacatacag 1921 ggctgtggtc aaaatcgtac ggacatctga ccaagtcgca aatttctgcc gccgagtctg 1981 tgccaagcta gagtgctgtc caaatttgtt tagtcctgtg ctgatatctg aaaactgccc 2041 agagaactgc tccattcata ccaaaaccaa atacacctat tactatggaa agagaaagaa 2101 gatctccaag ccccccatcg gggaaagcaa ccccgacagc ggacacccca aacccgccag 2161 gcggaggaag cgacggaaat ccattttcgt gcagaagaaa cggaggtctt ctgccgtgga 2221 cttcaccgcg ggctcggggg aggaaagtga agaggaggac gctgacgcca tggacgatga 2281 caccgccagt gaggagaccg gctccgagct ccgggatgac cagacggaca cctcgtcggc 2341 ggaggtgccc tcggcccggc cccggagggc cgtcaccctg cggagcggct cagagcccgt 2401 gcgccggcca cccccagaga ggacacgaag gggccgcggg gcgccggctg cctcctcagc 2461 agaggaaggg gagaagtgcc cgccgaccaa gcccgagggg acagaggaca cgaaacagga 2521 ggaggaggag agactggttc tggagagcaa cccgttggag tggacggtca ccgacgtggt 2581 gaggttcatt aagctgacag actgtgcccc cttggccaag atatttcagg agcaggatat 2641 tgacggccaa gcactcctgc ttctgaccct tccgacggtg caggagtgca tggagctgaa 2701 gctggggcct gccatcaagt tatgccacca gatcgagaga gtcaaagtgg ctttctacgc 2761 ccagtacgcc aactgagtct gccctcggga ggtggcccat tattgctggg atgcggtgtt 2821 ggtaaaggtt tccaggactg aaactttgat tttccgggat atgttaaatg gtacagccac 2881 taagtatcac cagaaaacca gaagcccagg atcttctgcc tccgccagcc tgtgagctgt 2941 ttccatgttt tcaaagcaca gcagcagtcg cttctgggga gtgccagtta aagtcatgca 3001 tcagaccctg ccagacgtgg gcctgcttct tggctcaccc acgttttgcc tttctcctgc 3061 cccaaatcag gcagctccct tggagcaggg tttcctcaga tgaggactgc attctttgaa 3121 aacaaagaat gtcgccaagg aagaaacctc acgccatgct gtagtgtttc ctgtaatcac 3181 acgagcacat ttatatatgc agtttcccat ggataggcgt gtgaccctgg ttgagtggca 3241 cttgcggttt catcttggtg gcaactcctt tgcaatgcag ctggcagcga catccttata 3301 aaaacatgtg ctaaagctct gtcctctgtt agaggtgcct tttaggaata cggggagtga 3361 aggaaggccg gcaggcatct ccatgcaact agatggtttg tttgtttgtt tgtttgtttg 3421 ttgttcattt tgttgtgttt tttgagacag ggtcttgctc tgtcgcccag gttgtaatgc 3481 agtggcgcaa tctcagctca ctgcaacctc tctctcccgg gttcaagtga ttctcctgcc 3541 tcagcctccc aagtagctgg gattacaggc acccaccacc atgcctggct aatttttgta 3601 tttttggtag agacagggtt tcaccatgtt ggtcaggcta gtcttgaact cccaacctca 3661 agtgatctgc ccgcctcggc ctcccaacgt gctgggatta caggtgtgag ccactacgcc 3721 ccggcccaac tggatggttt ttgattgaag cctagaacat ctgtagagac aaactctacc 3781 cagtcttttc tagaccctca actatctcca gtgttgttgt ttaatcgtag ccggatcagg 3841 gagtgagtct tttaggcaaa tgttggatta tatatcaaag gaaaagctta gtttcagaga 3901 ggaggaaggg aaagagatgt gagggaagca tttcatcaac cagctacgtc ccccttagaa 3961 ggatcactgc agcaggtcac cgagcaggag tccctctgag cgtcccttct gtctcgttct 4021 gccctagctg gcagcatatg aaccaggcat gatgcagcag gagcagtgaa tctggagtca 4081 gccacttggc accctggttt cgctgagaac aaactctgag atcttgggtg acttctcatc 4141 actctggacc tccattcctg tgaagtgaca ggtgtggacc ctgagggtgc ggtggtgagc 4201 acactgtctc ctgctggcat tcaccccact catgctggaa aggaagatcc agatcgtaca 4261 aaaattagaa aaagaaagaa taagaagggt ctggtcccag ttctgactcg gccattctta 4321 cagctctttc tggctttgag tttgcttgtg gaatttcctg ggcagttgtg ttaaatccgc 4381 caggtcacgt gcagacaaag ctgtggctgc gagagttggc tggcctcttg gaccagaagc 4441 catctccata tcctcatgag cgattccata tctccactca gaccctgtgg actacagtgt 4501 tccgctgtgg tggctgccaa gatgccttct taaacttatg caaggaaacc aaaccctccc 4561 acagttccca agcagacact ggaagcagag gcttctcacc cttcctgctt tttcaccaca 4621 atcaccttga gctcgtccct tggactagag tctccacagt tccagtaaaa ttctgcggtg 4681 ggctgatgag ctgcttgcat ttctgtgaca tttccagata tgattctcag tgggattttg 4741 gaaactttga ttgctcaagc tcacccttct taacattctg taatggttac agatgagaat 4801 ggaaaacaca tattttatgg atgaggcgtt ttggtctccc ctgcagtcga tttctagaat 4861 caagttttag agttcggctg atgcatctgc ctggggacct cagatgggag gagtgtgtca 4921 gttgtacccc gacagaaatg tctctgggat ctgtggctgg cttgccccgg gcatctctcc 4981 tttaagctca agttttgaac tctctgcggt tttccacccc tgccttctca gccacatgct 5041 tttggcctta aacgctcagt cttgtggagt tcaactctgt caaacgattg gaaagggcat 5101 ccatttccag atctttggca ttttccccgc gctgactctt tgatgatcct tcactgtggc 5161 cttttcaagc tcagctgttc ctgttgtatt tgagacgagg gtgagggaat gtggtggcca 5221 caaaagaaca gggacttgca gcacaaatgt cacttctgtc tcccttttca gtggtagcac 5281 ggaggaggag gtgctgcgtt ggagggaggg gatcctccag gagctctctg gagcccatct 5341 aggaagctag agtgtgtggc ccgccaggag ctcaggaagg atacagccac tgtcgcaggg 5401 gaaagtgttt gcttcccgtg gagccaagcg cccaagactc tccgtatcct tcaccctgac 5461 agtttaactt cagcgtttct ctgtgcagtt gcggtcacca tgggtgagca ctgtctgtgc 5521 acgtgccagg gaggagatgg ctgggaccac tgcacaggag ggcgcagcct ggcgtcgcca 5581 tgaaagttgt ctctgtgcca tctctccggt ccttgaggag agcccagaaa gattttagga 5641 cccaggaggt gcttttcctc cagctgttgc cagtgtcctt ctgagcctgg attctccggg 5701 gatttccgtc gtggtggatg gacttcacat cagcagcagt tctggtacag aattgtaatg 5761 tgttttcatt tctctgtagg attcacctct caccagcgtc tgtcttaaag gtagggccaa 5821 tttcatggag catttttctg tgtgtgtcct tgttgctttt gccagaaaaa gtggatttga 5881 catgcgtgcc ccgatgccac catagcccct aggccaacaa tgtcatggtc taaacaccaa 5941 aaagtgatgc cccgcattcc ttccctggat ggtaccgttt cttctccgtc tctctttgat 6001 gattctttgg gaccaaagtc ctctccttag tgcgcctact tcctgtgggc atcatgccac 6061 ttggaactta ttggaactgg cccgggagac tctgcagtct gcgccgtttg aaaaccctga 6121 gaaagagatg ccacctcaac ttgaatcatg acagcccatc gctcagtctc accctaaact 6181 catggagctt gtttcagctc ctcacttctt gactgtattt gtactatgtt gaaaaaatat 6241 cctgtccaca aagacataag cctaacaacc tagaaaaaca acagggtact actggcatta 6301 cagaacttct ttgcctttca aaacaaaagc aaaacacagt gaacttcacc acggagctgc 6361 acagcgtggg gaactcatcc atcactttca aaattagagt catttgatcc aagttggagt 6421 cagacacagt atttgagctg cacggcttct gggttctccc accttatttg atcatattcg 6481 aaagattatt tcctgtgttt gctttgattt gttcctcagt acattaaaat gatccacacc 6541 ttgaacactg ccctctctag aaggttgatt ttgatcagcc ttttgaagat gggtgtcgtt 6601 tccctaactt atctcacaga attttgagtg ttgtatttgg caagttctga gatttgcctt 6661 ctgtcttatg ccaaacaccc ctttctaaga gctgtccccg cttagtttta gaagtactag 6721 gggttttcat acttatttta tagaacaccc atttatattt atttctgtat atagaactaa 6781 aaaaaacagt agtgttaaaa atctttgttg tggtttgagc atctttgctg cttttggatt 6841 gagatggcga atcaaggctt cacttcctct ctcttctgtc tttagaaagc tgtgatcgtg 6901 cgtgcaatta tttgaaaggc aacatagtca attaagaaac ctgtagttgt taaggaagaa 6961 attgttggca agatatccat actgcccata tctcgttggt gcaataatta aatagcaaag 7021 gaaatctgta ttggcaacta ttataattca ataattcttt tgtttactgc ccttttctgt 7081 tcaagaattt tctggaaatt actccctttc acatggttga actcttaagt tgaccagttc 7141 tcatagctct atcactagaa tggtttgcag ataccccaaa catactatga taaaatcaaa 7201 ttgtgctact tttgacccat gtaatttacc taaaagttgt aattgctgac agagtactgc 7261 cttgaatttt ggtttaaaac ctctctagtt tcaatgacaa gtaacaactc aaataattcc 7321 atattgtttg aggaagaggc cataatcctt ctgaattgtt ggcactaagt aatgggattt 7381 ggcccagtaa gtatgacggt cgtgtcgcct aaccaacgca gagcagtgct ttttgtgtgg 7441 ctgaagcgat gtgctgacga aaaaaggaaa attctaggac aatcgttggc taaaaatcac 7501 cttaggatga aaaatttgag gcaaattttt ttaaatgaca gaaaaagata atcatctcac 7561 ttgcttgaaa caggagccag catgatctct ggaagcatca actatccctc gtcgtgattg 7621 ttgaaagctc tttcactgtt ttgcattcta gtttgaatag tttgtattga aattggattc 7681 ctatcttgtg tatgtttttg gtgcgtaaaa gggaaaaatt ggtgtcatta cttttgaaat 7741 ttgcaggacg aagggcatgc ttttggtttg ctgtaagatt gtattctgta tatatgtttt 7801 catgtaaata aatgaaaatc tatatcagag ttatatttta atttttattc taaatgaaaa 7861 aaaccctttt tacttcaaaa aaattgtaag ccacattgtt aataaagtaa aaataaattc 7921 ta

By “Smoc1 polypeptide” (SPARC related modular calcium binding 1) is meant a protein having at least about 85% amino acid identity to the sequence provided at NCBI Reference Sequence: NP_001030024, or a fragment thereof. An exemplary Smoc1 amino acid sequence is provided below (SEQ ID NO: 14):

1 mlparcarll tphlllvlvq lsparghrtt gprflisdrd pqcnlhcsrt qpkpicasdg 61 rsyesmceyq rakcrdptlg vvhrgrckda gqskcrlera qaleqakkpq eavfvpecge 121 dgsftqvqch tytgycwcvt pdgkpisgss vqnktpvcsg svtdkplsqg nsgrkddgsk 181 ptptmetqpv fdgdeitapt lwikhlvikd sklnntnirn sekvyscdqe rqsaleeaqq 241 npregivipe capgglykpv qchqstgycw cvlvdtgrpl pgtstryvmp scesdarakt 301 teaddpfkdr elpgcpegkk mefitsllda lttdmvqain saaptgggrf sepdpshtle 361 ervvhwyfsq ldsnssndin kremkpfkry vkkkakpkkc arrftdycdl nkdkvislpe 421 lkgclgvske vgrlv

By “Smoc1 polynucleotide” is meant a nucleic acid molecule encoding a Smoc1 polypeptide. An exemplary Smoc1 polynucleotide sequence is provided at XM_005267995.1, which is reproduced below (SEQ ID NO: 15):

1 ataacgggaa ttcccatggc ccgggctcag gcgtccaacc tgctgccgcc tgggccccgc 61 cgagcggagc tagcgccgcg cgcagagcac acgctcgcgc tccagctccc ctcctgcgcg 121 gttcatgact gtgtcccctg accgcagcct ctgcgagccc ccgccgcagg accacggccc 181 gctccccgcc gccgcgaggg ccccgagcga aggaaggaag ggaggcgcgc tgtgcgcccc 241 gcggagcccg cgaaccccgc tcgctgccgg ctgcccagcc tggctggcac catgctgccc 301 gcgcgctgcg cccgcctgct cacgccccac ttgctgctgg tgttggtgca gctgtcccct 361 gctcgcggcc accgcaccac aggccccagg tttctaataa gtgaccgtga cccacagtgc 421 aacctccact gctccaggac tcaacccaaa cccatctgtg cctctgatgg caggtcctac 481 gagtccatgt gtgagtacca gcgagccaag tgccgagacc cgaccctggg cgtggtgcat 541 cgaggtagat gcaaagatgc tggccagagc aagtgtcgcc tggagcgggc tcaagccctg 601 gagcaagcca agaagcctca ggaagctgtg tttgtcccag agtgtggcga ggatggctcc 661 tttacccagg tgcagtgcca tacttacact gggtactgct ggtgtgtcac cccggatggg 721 aagcccatca gtggctcttc tgtgcagaat aaaactcctg tatgttcagg ttcagtcacc 781 gacaagccct tgagccaggg taactcagga aggaaagtct cctttcgatt ctttttaacc 841 ctcaattcag atgacgggtc taagccgaca cccacgatgg agacccagcc ggtgttcgat 901 ggagatgaaa tcacagcccc aactctatgg attaaacact tggtgatcaa ggactccaaa 961 ctgaacaaca ccaacataag aaattcagag aaagtctatt cgtgtgacca ggagaggcag 1021 agtgccctgg aagaggccca gcagaatccc cgtgagggta ttgtcatccc tgaatgtgcc 1081 cctgggggac tctataagcc agtgcaatgc caccagtcca ctggctactg ctggtgtgtg 1141 ctggtggaca cagggcgccc gctgcctggg acctccacac gctacgtgat gcccagttgt 1201 gagagcgacg ccagggccaa gactacagag gcggatgacc ccttcaagga cagggagcta 1261 ccaggctgtc cagaagggaa gaaaatggag tttatcacca gcctactgga tgctctcacc 1321 actgacatgg ttcaggccat taactcagca gcgcccactg gaggtgggag gttctcagag 1381 ccagacccca gccacaccct ggaggagcgg gtagtgcact ggtatttcag ccagctggac 1441 agcaatagca gcaacgacat taacaagcgg gagatgaagc ccttcaagcg ctacgtgaag 1501 aagaaagcca agcccaagaa atgtgcccgg cgtttcaccg actactgtga cctgaacaaa 1561 gacaaggtca tttcactgcc tgagctgaag ggctgcctgg gtgttagcaa agaagtagga 1621 cgcctcgtct aaggagcaga aaacccaagg gcaggtggag agtccaggga ggcaggatgg 1681 atcaccagac acctaacctt cagcgttgcc catggccctg ccacatcccg tgtaacataa 1741 gtggtgccca ccatgtttgc acttttaata actcttactt gcgtgttttg tttttggttt 1801 cattttaaaa caccaatatc taataccaca gtgggaaaag gaaagggaag aaagacttta 1861 ttctctctct tattgtaagt ttttggatct gctactgaca acttttagag ggttttgggg 1921 gggtggggga gggtgttgtt ggggctgaga agaaagagat ttatatgctg tatataaata 1981 tatatgtaaa ttgtatagtt cttttgtaca ggcattggca ttgctgtttg tttatttctc 2041 tccctctgcc tgctgtgggt ggtgggcact ctggacacat agtccagctt tctaaaatcc 2101 aggactctat cctgggccta ctaaacttct gtttggagac tgacccttgt gtataaagac 2161 gggagtcctg caattgtact gcggactcca cgagttcttt tctggtggga ggactatatt 2221 gccccatgcc attagttgtc aaaattgata agtcacttgg ctctcggcct tgtccaggga 2281 ggttgggcta aggagagatg gaaactgccc tgggagagga agggagtcca gatcccatga 2341 atagcccaca caggtaccgg ctctcagagg gtccgtgcat tcctgctctc cggaccccca 2401 aagggcccag cattggtggg tgcaccagta tcttagtgac cctcggagca aattatccac 2461 aaaggatttg cattacgtca ctcgaaacgt tttcatccat gcttagcatc tactctgtat 2521 aacgcatgag aggggaggca aagaagaaaa agacacacag aagggccttt aaaaaagtag 2581 atatttaata tctaagcagg ggaggggaca ggacagaaag cctgcactga ggggtgcggt 2641 gccaacaggg aaactcttca cctccctgca aacctaccag tgaggctccc agagacgcag 2701 ctgtctcagt gccaggggca gattgggtgt gacctctcca ctcctccatc tcctgctgtt 2761 gtcctagtgg ctatcacagg cctgggtggg tgggttgggg gaggtgtcag tcaccttgtt 2821 ggtaacacta aagttgtttt gttggttttt taaaaaccca atactgaggt tcttcctgtt 2881 ccctcaagtt ttcttatggg cttccaggct ttaagctaat tccagaagta aaactgatct 2941 tgggtttcct attctgcctc ccctagaagg gcaggggtga taacccagct acagggaaat 3001 cccggcccag ctttccacag gcatcacagg catcttccgc ggattctagg gtgggctgcc 3061 cagccttctg gtctgaggcg cagctccctc tgcccaggtg ctgtgcctat tcaagtggcc 3121 ttcaggcaga gcagcaagtg gcccttagcg ccccttccca taagcagctg tggtggcagt 3181 gagggaggtt gggtagccct ggactggtcc cctcctcaga tcacccttgc aaatctggcc 3241 tcatcttgta ttccaacccg acatccctaa aagtacctcc acccgttccg ggtctggaag 3301 gcgttggcac cacaagcact gtccctgtgg gaggagcaca accttctcgg gacaggatct 3361 gatggggtct tgggctaaag gaggtccctg ctgtcctgga gaaagtccta gaggttatct 3421 caggaatgac tggtggccct gccccaacgt ggaaaggtgg gaaggaagcc ttctcccatt 3481 agccccaatg agagaactca acgtgccgga gctgagtggg ccttgcacga gacactggcc 3541 ccactttcag gcctggagga agcatgcaca catggagacg gcgcctgcct gtagatgttt 3601 ggatcttcga gatctcccca ggcatcttgt ctcccacagg atcgtgtgtg taggtggtgt 3661 tgtgtggttt tcctttgtga aggagagagg gaaactattt gtagcttgtt ttataaaaaa 3721 taaaaaatgg gtaaatcttg

By “tri-methylated histone H3 at lysine 27 (H3K27me3)” is meant the trimethylation of lysine 27 on histone H3 protein subunit. The H3K27me3 modification is generally associated with gene repression.

By “agent” is meant a peptide, nucleic acid molecule, or small compound.

By “allele” is meant one of two or more alternative forms of a gene that are found at the same place on a chromosome.

By “alteration” is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.”

By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

In this disclosure, “comprises,” “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of” or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

By “disease” is meant any condition or disorder that damages, or interferes with the normal function of a cell, tissue, or organ. Examples of disorders include those associated with undesirable repression of an allele by H3K27me3-dependent imprinting. Microphthalmia exemplary disorder associated with H3K27me3-dependent imprinting relating to imprinting disorders.

By “DNA” is meant deoxyribonucleic acid. In various embodiments, the term DNA refers to genomic DNA, recombinant DNA, or cDNA. In particular embodiments, the DNA comprises a “target region.” DNA libraries contemplated herein include genomic DNA libraries, and cDNA libraries constructed from RNA, e.g., an RNA expression library. In various embodiments, the DNA libraries comprise one or more additional DNA sequences and/or tags.

By “effective amount” is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.

By “fragment” is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is free to varying degrees from components which normally accompany it as found in its native state. “Isolate” denotes a degree of separation from original source or surroundings. “Purify” denotes a degree of separation that is higher than isolation. A “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an “isolated polypeptide” is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

By “reference” is meant a standard or control condition.

A “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having “substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. By “hybridize” is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C., more preferably of at least about 37° C., and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C. in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100·mu·g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C., more preferably of at least about 42° C., and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

By “Somatic Cell Nuclear Transfer” or “SCNT” is meant the transfer of a donor nucleus from a somatic cell into an enucleated oocyte. The process can be used in either reproductive or therapeutic cloning. By “subject” is meant a mammal, including, but not limited to, a human or non-human mammal, such as an agriculturally significant mammal (e.g., bovine, equine, ovine), a pet (e.g., canine, feline), or a rare or endangered mammal (e.g., panda).

As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural (i.e., at least one). By way of example, “an element” means one element or more than one element.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-FIG. 1C shows allelic DNase I hypersensitive sites (DHSs) in zygotes mark allelic gene expression at ZGA.

FIG. 1A provides a schematic for identifying parental allele-specific DHSs in zygotes. IVF, in vitro fertilization. PN, pronucleus. FIG. 1B depicts a heat map showing bi-allelic, paternal allele-specific (Ps-DHSs), and maternal allele-specific DHSs (Ms-DHSs) in zygotes. Each row represents liDNase-seq (low-input DNase-seq) signal intensity at a DHS±5 kb.

FIG. 1C provides representative androgenesis (AG)- and gynogenesis (GG)-specific differentially expressed genes (DEGs) harboring allelic promoter DHSs in zygotes. Upper panels are genome browser views of DHSs in paternal and maternal pronuclei with biological duplicates. The DHS signal intensity and the genomic length of each view (kb) are indicated at the upper left and the bottom of each panel, respectively. Lower graphs are gene expression levels in AG, GG and a-amanitin-treated (Ama) 2-cell embryos. Error bar, standard deviation of biological duplicates. Note that GG-specific expression of Akap1 and Isl2 is evident after subtraction of maternal pool transcripts.

FIG. 2A-FIG. 2H shows identification of parental allelic DHSs, related to FIG. 1 . FIG. 2A provides scatter plots showing the correlation of DHSs between three biological replicates in paternal and maternal pronuclei (PN).

FIG. 2B provides a scatter plot showing bi-allelic DHSs (upper-right), Ps-DHSs (upper-middle left), and Ms-DHSs (left). The cutoffs used to define these DHS groups are indicated.

FIG. 2C provides averaged DHS signals of Ps-DHSs and Ms-DHSs within ±5 kb around DHSs.

FIG. 2D provides genomic distribution of DHSs. Promoters represent ±1 kb around transcriptional start sites (TSSs). ‘Random’ indicates the percentages of each genomic element of the mouse genome. FIG. 2E. provides percentages of DHSs located at CpG islands (CGIs). Promoters represent ±1 kb around TSSs. The genomic locations of CGIs were described in Kobayashi et al., 2012.

FIG. 2F provides a genome browser view of Ps-DHSs at imprinting control regions (ICRs) of representative imprinted genes. The genomic locations of ICRs were referred in Kobayashi et al., 2012.

FIG. 2G provides a list of genes harboring promoter Ps-DHSs or Ms-DHSs in zygotes.

FIG. 2H provides a genome browser view of representative allelic DHSs at gene promoters not previously known to be imprinted.

FIG. 3A-FIG. 3F shows correlation between allelic ZGA in two-cell embryos and allelic DHSs in zygotes. The experimental scheme, RNA-seq reproducibility and analysis scheme are related to FIG. 1

FIG. 3A provides a schematic for identifying parental allele-specific gene expression at ZGA. Androgenetic (AG) embryos and gynogenetic (GG) embryos were produced by pronuclear transfer. AG 2-cell embryos contain paternally-expressed nascent transcripts and maternally-stored transcripts. GG 2-cell embryos contain maternally-expressed nascent transcripts and maternally-stored transcripts. α-amanitin-treated (Ama) 2-cell embryos contain maternally-stored transcripts only.

FIG. 3B provides a scatter plot showing the correlation between biological duplicate of 2-cell RNA-seq samples.

FIG. 3C provides a flowchart for avoiding maternally-stored transcripts and identifying nascent allelic transcripts at ZGA.

FIG. 3D provides a scatterplot of nascent transcripts in AG and GG 2-cell embryos. For each gene, the FPKM value in Ama embryos was subtracted from that in AG and GG embryos, respectively. AG- and GG-specific differentially expressed genes (DEGs) (FC>10) are indicated either below the dashed-line or above the dashed-line, respectively. Known imprinted genes are indicated with their associated name below the dashed-line.

FIG. 3E and FIG. 3F provide a scatterplot showing DHS allelic bias at promoters (±0.5 kb at TSS) of androgenesis- (FIG. 3E) and gynogenesis- (FIG. 3F) specific differentially expressed genes (DEGs). FC>2 was considered as ‘bias’ dark gray in FIGS. 3E and 3F.

FIG. 4A-FIG. 4H shows zygotic Ms-DHSs are inherited from oocyte DHSs, related to FIG. 6 .

FIG. 4A provides a scatter plot showing the correlation between three biological replicates of liDNase-seq for germinal vesicle (GV) nuclei isolated from fully-grown oocytes.

FIG. 4B provides a genome browser view of sperm DHSs that are passed on to paternal PNs of zygotes. The nearest gene names are indicated at the top of each panel.

FIG. 4C provides a heat map showing Ps-DHSs. Each row represents liDNase-seq signal intensity at a DHS±5 kb. Note that Ps-DHSs are largely absent in both sperm or oocytes.

FIG. 4D provides a genome browser view of representative Ps-DHSs.

FIG. 4E provides a heat map showing Ms-DHSs. Note that Ms-DHSs are mostly already present in oocytes.

FIG. 4F provides a genome browser view of representative Ms-DHSs.

FIG. 4G provides a heat map showing biallelic DHSs.

FIG. 4H provides a genome browser view of representative biallelic DHSs.

FIG. 5A-FIG. 5J shows distinct epigenetic features of Kdm6b- and Kdm4d-affected Ps-DHSs, related to FIG. 6 .

FIG. 5A provides a pie chart showing percentages of Ps-DHSs that overlap (black) or associated (gray) with oocyte-gametic differentially methylated regions (gDMRs) within ±100 kb. Oocyte gDMR was defined by >80% methylation in oocytes and <20% methylation in sperm.

FIG. 5B provides a pie chart showing the percentages of Ps-DHSs organized based on their oocyte DNA methylation levels.

FIG. 5C provides boxplots showing the H3K27me3 signal levels at Ps-DHSs±1 kb in gametes (left panel) and zygotes (right panel). Ps-DHSs were divided into oocyte DNA hypomethylated (0-20%, n=296) and hypermethylated groups (80-100%, n=305). Middle lines in the boxes represent the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively.

FIG. 5D provides representative images of Kdm6b- or Kdm4d-injected zygotes stained with anti-Flag antibody, using non-injected zygotes as negative controls.

FIG. 5E provides representative images of zygotes stained with anti-H3K27me3 antibody. M, maternal pronucleus. P, paternal pronucleus. The bar graph on the right represents relative immunostaining signal intensity of maternal pronuclei. The averaged signal of non-injected zygotes was set as 1.0. The total numbers of embryos examined were 8 (No injection), 13 (Kdm6b^(WT)), and 10 (Kdm6b^(MUT)). Error bars indicate SD. ***, p<0.001 (two-tailed Student t-test). N.S, statistically not significant.

FIG. 5F provides representative images of zygotes stained with anti-H3K9me3 antibody. The bar graph on right represents relative immunostaining signal intensity in the maternal pronuclei. The averaged signal of non-injected zygotes was set as 1.0. The total numbers of embryos examined were 5 (no-inject), 5 (Kdm4d^(WT)), and 7 (Kdm4d^(MUT)). Error bars indicate SD. ***, p<0.001 (two-tailed Student t-test). N.S, statistically not significant.

FIG. 5G provides a scatter plot showing the correlation between biological duplicates of liDNase-seq for maternal (Mat) and paternal pronuclei (Pat) of Kdm6b^(WT)- and Kdm6b^(MUT)-injected zygotes.

FIG. 5H provides a scatter plot showing the correlation between biological duplicates of liDNase-seq for maternal (Mat) and paternal pronuclei (Pat) of Kdm4d^(WT)- and Kdm4d^(MUT)-injected zygotes.

FIG. 5I provides a genome browser view of representative Ps-DHSs affected by Kdm4d^(WT).

FIG. 5J provides a boxplot showing H3K27me3 signals at Kdm6b- or Kdm4d-affected Ps-DHSs±1 kb in gametes (left panel) and zygotes (right panel). Middle lines in the boxes indicate the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively.

FIG. 6A-FIG. 6D shows oocyte-specific H3K27me3 prevents maternal chromatin accessibility at DNA hypomethylated regions.

FIG. 6A provides a schematic for studying the role of histone methylations in maternal chromatin inaccessibility.

FIG. 6B provides a heat map showing the allelic bias at Ps-DHSs in Kdm6b- or Kdm4d-injected zygotes.

FIG. 6C provides a genome browser view of representative Ps-DHSs affected by Kdm6b^(WT).

FIG. 6D provides pie charts showing Kdm6b- or Kdm4d-affected Ps-DHSs organized based on their oocyte DNA methylation levels.

FIG. 7A-FIG. 7D shows genes with H3K27me3-marked AG-DHSs are paternally expressed in morula embryos.

FIG. 7A provides a schematic for identifying parental allele-specific DHSs in morula embryos.

FIG. 7B provides a heat map showing AG-specific (AG-DHSs) and GG-specific DHSs (GG-DHSs) in morula embryos. Each row represents liDNase-seq signal intensity at a DHS 5 kb.

FIG. 7C provides a scatterplot showing allelic enrichment of H3K27me3 ChIP-seq signal at AG-DHSs±1 kb in inner cell mass (ICM) of blastocyst embryos. AG-DHSs with [RPM>0.5, FC(Mat/Pat)>2] were considered to harbor maternal allele-biased H3K27me3 (dark gray dots).

FIG. 7D provides a heat map showing parental allele-specific gene expression of putative H3K27me3-dependent imprinted genes. Genes expressed in AG morula embryos (RPKM>0.5) are shown. The left column represents the ratio of AG/GG gene expression. The two right columns represent relative gene expression in hybrid morula embryos. B×C; B6/CAST. C×B; CAST/B6. The 4 known non-canonical imprinted genes are indicated in bold. White boxes indicate ‘not determined (N.D.)’ due to lack of SNP reads (<20 reads).

FIG. 8A-FIG. 8D shows androgenetic (AG)- and gynogenetic (GG)-specific DHSs in morula embryos, related to FIG. 7 .

FIG. 8A provides a scatter plot showing the correlation between biological duplicates of liDNase-seq for AG and GG morula embryos.

FIG. 8B provides averaged SNP-tracked liDNase-seq signal intensity of paternal and maternal alleles in hybrid morula embryos. The data were obtained from morula embryos of a BDF1 and JF1 cross. Plots from the biological duplicates (e.g. BDF1_1 and BDF1_2) are shown. Note that paternal (JF1), but not maternal (BDF1), SNP reads are enriched in AG-DHSs (left panel), while neither SNP reads are enriched in GG-DHSs (right panel).

FIG. 8C provides a genome browser view of DHSs at known imprinting control regions (ICRs).

FIG. 8D provides a pie chart showing AG-DHSs grouped based on their oocyte DNA methylation levels.

FIG. 9A-FIG. 9D shows allelic gene expression in morula embryos, related to FIG. 7 .

FIG. 9A provides a scatter plot showing the correlation between biological duplicates of RNA-seq samples.

FIG. 9B provides a scatterplot of gene expression levels in AG- and GG morula embryos. AG- and GG-specific differentially expressed genes (DEGs) (FC>10) are indicated either below the dashed-line or above the dashed-line, respectively. Paternally-expressed known imprinted genes are below the dashed-line and include their associated gene names. A maternally-expressed known gene, Meg3, is indicated above the dashed-line.

FIG. 9C provides genome browser views of allelic H3K27me3 levels in non-canonical imprinted genes. Sp; sperm. Oo; MII-stage oocyte. ICM; inner cell mass of blastocysts. Paternal (Pat) and maternal (Mat) allele signals in 1-cell and ICM were based on SNP analyses.

FIG. 9D provides genome browser views of allelic H3K27me3 levels in representative canonical imprinted genes. Known ICRs are indicated at the bottom of each canonical imprinted gene.

FIG. 10A-FIG. 10E shows maternal H3K27me3 serves as an imprinting mark.

FIG. 10A provides a schematic for studying the role of H3K27me3 in maternal allele repression. Kdm6b^(MUT)-injected parthenogenetic (PG) embryos were used as a negative control.

FIG. 10B provides relative gene expression levels (log scale) of putative H3K27me3-dependent imprinted genes. Shown are genes expressed in AG morula embryos (RPKM>0.5) and significantly derepressed by Kdm6b^(WT). The expression level of gynogenetic (GG) morula embryos was set as 1. The genes are ordered by statistical significance (p-values by DEseq) between Kdm6b^(WT) and Kdm6b^(MUT) samples. Arrows indicate known non-canonical imprinted genes.

FIG. 10C provides a heat map showing parental allele-specific gene expression of putative H3K27me3-dependent imprinted genes in Kdm6b^(WT)- and Kdm6b^(MUT)-injected hybrid morula embryos. Among the 28 genes listed in FIG. 3 d , those with >10 SNP reads in both samples are shown. Known non-canonical imprinted genes are indicated in bold. Allelic expression levels of representative canonical imprinted genes are shown at the bottom.

FIG. 10D provides a heat map showing the levels of chromatin accessibility at AG-DHSs in Kdm6b^(WT)- and Kdm6b^(MUT)-injected morula PG embryos. The DHS signal intensity in AG embryos was set as 100%. AG-DHSs are ordered by Δ(Kdm6b^(WT)−Kdm6b^(MUT)). Known imprinted genes are indicated at right, with non-canonical imprinted genes shown in at the upper right side of the panel in light gray font.

FIG. 10E provides a genome browser view of gain-of-accessibility at AG-DHSs of putative H3K27me3-dependent imprinted genes.

FIG. 11A-FIG. 11G shows the effect of Kdm6b mRNA injection on maternal allele expression and accessibility, related to FIG. 10 .

FIG. 11A provides a developmental ratio of Kdm6b^(WT)- and Kdm6^(MUT)-injected parthenogenetic (PG) embryos. The total embryo numbers examined were 60 (WT) and 58 (MUT).

FIG. 11B provides a scatter plot showing the correlation between biological duplicates of RNA-seq for Kdm6b^(WT)- and Kdm6^(MUT)-injected PG embryos.

FIG. 11C provides relative gene expression levels of canonical imprinted genes that are expressed in AG morula embryos (RPKM>0.5). Note that none are derepressed by Kdm6b^(WT) injection.

FIG. 11D provides a scatter plot showing the correlation between biological duplicates of liDNase-seq for Kdm6b^(WT)- and Kdm6b^(MUT)-injected PG embryos.

FIG. 11E and FIG. 11F provide wide genome browser views of non-canonical (e) and canonical imprinted genes (f). The arrowheads indicate AG-DHSs at which chromatin accessibility is gained in Kdm6b^(WT)-injected PG embryos (shown in FIG. 4 e ). Known imprinting control regions (ICRs) are indicated above each panel of canonical imprinted genes (f).

FIG. 11G provides a genome browser view of AG-DHSs of representative canonical imprinted genes.

FIG. 12A-FIG. 12E shows cell lineage-specific dynamics of H3K27me3-dependent genomic imprinting.

FIG. 12A provides a heat map showing parental allele-specific gene expression of putative H3K27me3-dependent imprinted genes in hybrid blastocyst embryos. B×C; B6/CAST. C×B; CAST/B6. Known non-canonical imprinted genes are indicated in bold in panels a-d. The grayscale scheme in panels a-d follows FIG. 7 d.

FIG. 12B provides a heat map showing androgenesis/gynogenesis (AG/GG) relative expression of putative H3K27me3-dependent imprinted genes in ICM and TE of blastocyst embryos. Arrows indicate genes showing a milder level of AG-bias in ICM when compared to TE. White boxes indicate ‘not determined’ due to low gene expression levels (RPKM<0.5).

FIG. 12C provides a heat map showing parental allele-specific gene expression of putative H3K27me3-dependent imprinted genes in epiblast (EPI), visceral endoderm (VE), and extra-embryonic ectoderm (EXE) of E6.5 embryos. Genes with >20 SNP reads in both reciprocal crosses are shown. B×P; B6/PWK. P×B; PWK/B6. Arrowheads indicate genes showing imprinted expression.

FIG. 12D provides a heat map showing parental allele-specific gene expression of putative H3K27me3-dependent imprinted genes in pure fetus-derived E9.5 placenta cells. Genes with >20 SNP reads in both reciprocal crosses are shown. Arrowheads genes showing imprinted expression.

FIG. 12E provides a model illustrating the fate of H3K27me3-dependent genomic imprinting during development.

FIG. 13A-FIG. 13E shows genomic imprinting in E6.5 embryos, related to FIG. 12 .

FIG. 13A provides expression levels of marker genes for TE (Cdx2) and ICM (Sox2) in the samples.

FIG. 13B provides scatter plot showing the correlation between biological duplicates of the E6.5 epiblast (EPI), visceral endoderm (VE), and extra-embryonic ectoderm (EXE) RNA-seq samples from both B6×PWK and PWK×B6 crosses.

FIG. 13C provides bar graphs showing the expression levels of marker genes for epiblast (Pou5f1 and Nanog), extra-embryonic ectoderm (Elf5 and Gata3), and visceral endoderm genes (Gata6 and Gata4) in the samples.

FIG. 13D provides a heat map showing paternally-expressed genes (PEGs) and maternally-expressed genes (MEGs) in epiblast, visceral endoderm, and extra-embryonic ectoderm of E6.5 embryos. B×P; B6/PWK. P×B; PWK/B6. All genes showing parental allele-specific expression (FC>2 in both B×P and P×B) in each sample are shown. Genes not previously known to be imprinted are indicated in bold.

FIG. 13E provides a genome browser view of RNA-seq data of newly identified imprinted genes. D7Ertd715e and Smoc1 are paternally expressed, and Mas1 is maternally expressed. EXE, extra-embryonic ectoderm. VE, visceral endoderm.

FIG. 14A-FIG. 14C shows sample preparation and quality verification, related to FIG. 12

FIG. 14A provides an experimental scheme of placenta cell purification. Sperm or oocytes were collected from B6^(GFP) mice, and in vitro fertilized with the counterparts collected from the PWK strain. Embryos were transplanted into surrogate mothers. The placentae were harvested at E9.5, and dissociated into single cells by trypsin treatment before FACS sorting of GFP-positive cells.

FIG. 14B provides a scatter plot showing the correlation between biological duplicates of RNA-seq samples.

FIG. 14C provides total numbers of the paternal and maternal SNP reads in the purified placental cells.

FIG. 15A and FIG. 15B show genomic imprinting in E9.5 placentae, related to FIG. 12 .

FIG. 15A provides a heat map showing paternally-expressed genes (PEGs) and maternally-expressed genes (MEGs) in E9.5 placentae. B×P; B6/PWK. P×B; PWK/B6. All genes exhibiting parental allele-specific expression (FC>2 in both B×P and P×B) are shown. Genes not previously known to be imprinted are indicated in bold.

FIG. 15B provides a genome browser view of RNA-seq data of newly identified imprinted genes. D7Ertd715e and Smoc1 are paternally expressed, and Cbx7 and Thbs2 are maternally expressed.

FIG. 16A and FIG. 16B show maternal H3K27me3 coats Xist and persists through preimplantation development.

FIG. 16A provides a genome browser view of the H3K27me3 enrichment in gametes and growing oocytes, as well as DNaseI-seq signals and DNA methylation levels in GV oocytes at the Xist locus. The top center bar indicates the maternal H3K27me3 domain coating Xist. The H3K27me3 ChIP-seq, DNaseI-seq, and DNA methylome datasets were from (Zheng et al., 2016), (Inoue et al., 2017), and (Kobayashi et al., 2012), respectively. Oo, MII oocyte. Sp, sperm. 7d and 14d indicate growing oocytes collected from 7- and 14-day old females, respectively. GV, fully-grown GV-stage oocytes collected from 8-week old females.

FIG. 16B provides a genome browser view of the allelic H3K27me3 in 1-cell, 2-cell, and blastocyst embryos at the Xist locus. The highlighted square indicates a computationally determined region where the maternal allele-biased enrichment of H3K27me3 is retained in blastocyst embryos. Mat, maternal chromatin. Pat, paternal chromatin. The H3K27me3 ChIP-seq datasets were from (Zheng et al., 2016).

FIG. 17A-FIG. 17D shows ectopic removal of H3K27me3 induces maternal Xist expression.

FIG. 17A provides an experimental scheme for addressing the role of H3K27me3 in maternal Xist repression during preimplantation development.

FIG. 17B provides representative images of Xist RNA FISH (top row, light grey) in Kdm6b-injected morula embryos. The gender of each embryo was assessed by simultaneous DNA FISH using a green fluorescent BAC probe containing the Rnf12 locus on the X chromosome (middle row, arrow).

FIG. 17C and FIG. 17D provide the ratio of blastomeres showing the indicated number of Xist RNA clouds in male (FIG. 17C) and female (FIG. 17D) morula embryos. Each bar represents an individual embryo. The numbers of embryos examined were 19 (Kdm6b^(WT)) and 35 (Kdm6b^(MUT)) males and 34 (Kdm6b^(WT)) and 35 (Kdm6b^(MUT)) females.

FIG. 18A-FIG. 18C shows ectopic removal of H3K27me3 induces maternal XCI.

FIG. 18A provides a box plot showing the relative expression of genes on individual maternal chromosomes between Kdm6b^(MUT)- and Kdm6b^(WT)-injected blastocysts. Genes with enough SNP reads (RPM>0.5) were analyzed. Middle lines in the boxes represent the medians. Box edges and whiskers indicate the 25th/75th and 2.5th/97.5th percentiles, respectively. ***, p<0.001 (Mann-Whitney-Wilcoxon Test).

FIG. 18B and FIG. 18C provide the relative expression levels of Xm-linked genes between Kdm6b^(WT) and Kdm6b^(MUT) injected blastocyst embryos. Each dot represents an individual gene showing enough SNP reads (RPM>0.5). Panel c shows known escapees, and panel b shows the rest of genes.

FIG. 19A and FIG. 19B show Kdm6b mRNA injection results in loss of H3K27me3 in a catalytic activity-dependent manner, related to FIG. 17 .

FIG. 19A provides representative images of zygotes stained with anti-H3K27me3 antibody. M, maternal pronucleus. P, paternal pronucleus.

FIG. 19B provides relative immunostaining signal intensity of maternal pronuclei. The averaged signal of non-injected zygotes was set as 1.0. The total numbers of embryos examined were 8 (No injection), 13 (Kdm6b^(WT)), and 10 (Kdm6b^(MUT)). Error bars indicate SD. ***, p<0.001 (two-tailed Student t-test). N.S, statistically not significant.

FIG. 20A-FIG. 20E shows ectopic removal of H3K9me3 does not induce maternal Xist expression, related to FIG. 17 .

FIG. 20A provides representative images of zygotes stained with anti-H3K9me3 antibody. M, maternal pronucleus. P, paternal pronucleus.

FIG. 20B provides relative immunostaining signal intensity in the maternal pronuclei. The averaged signal intensity of non-injected zygotes was set as 1.0. The total numbers of embryos examined were 5 (no injection), 5 (Kdm4d^(WT)), and 7 (Kdm4d^(MUT)). Error bars indicate SD. ***, p<0.001 (two-tailed Student t-test). N.S, statistically not significant.

FIG. 20C provides representative images of Xist RNA FISH (magenta) in Kdm4b-injected morula embryos. The gender of each embryo was assessed by simultaneous DNA FISH using a green fluorescent BAC probe containing the Rnf12 locus on the X chromosome (arrow).

FIG. 20D and FIG. 20E provide the ratio of blastomeres that show the indicated number of Xist RNA clouds in male (FIG. 20D) and female (FIG. 20E) morula embryos. Each bar represents an individual embryo. The numbers of embryos examined were 9 (Kdm4d^(WT)) and 12 (Kdm4d^(MUT)) males and 9 (Kdm4d^(WT)) and 15 (Kdm4d^(MUT)) females.

FIG. 21 provides a scatter plot showing the correlation between biological duplicate of RNA-seq samples, related to FIG. 18 .

DETAILED DESCRIPTION

The invention provides methods for activating a H3K27me3 silenced allele within an imprinting control region by contacting the silenced allele with an agent that removes H3K27me3 or with an agent that inhibits H3K27 trimethylation, thereby treating a H3K27me3-dependent imprinting associated disorder.

The invention is based, at least in part, on the discovery that maternal H3K27me3 acts as a DNA methylation-independent imprinting mechanism, and that H3K27me3 is the imprinting mark of Xist an X-linked long non-coding RNA, which functions in X-chromosome inactivation.

H3K27me3 is a DNA Methylation-Independent Imprinting Mechanism

Mammalian sperm and oocytes have different epigenetic landscapes and are organized in different fashion. Following fertilization, the initially distinct parental epigenomes become largely equalized with the exception of certain loci including imprinting control regions (ICRs). How parental chromatin becomes equalized and how ICRs escape from this reprogramming is largely unknown. Here parental allele-specific DNase I hypersensitive sites (DHSs) was characterized in mouse zygotes and morula embryos, and the epigenetic mechanisms underlying allelic DHSs was investigated. Integrated analyses of DNA methylome and H3K27me3 ChIP-seq data sets revealed 76 genes with paternal allele-specific DHSs that were devoid of DNA methylation, but harbored maternal allele-specific H3K27me3. Interestingly, these genes are paternally expressed in preimplantation embryos, and ectopic removal of H3K27me3 induced maternal allele expression. H3K27me3-dependent imprinting was largely lost in the embryonic cell lineage, but at least 5 genes maintained their imprinting in the extra-embryonic cell lineage. The 5 genes include all previously identified DNA methylation-independent imprinted autosomal genes. Thus, the results reported herein identified maternal H3K27me3 as a DNA methylation-independent imprinting mechanism.

Accordingly, the invention provides methods for relieving undesirable H3K27me3-dependent imprinting in a cell, including in the cell of a subject having an H3K27me3-dependent imprinting associated disorder. In one embodiment, such methods involve the use of an H3K27me3 selective methylase.

H3K27me3 is Important for X Chromosome Inactivation

In females of certain therian mammals including rodents, one of the two X chromosomes is inactivated to achieve gene dosage compensation. This phenomenon, called X chromosome inactivation (XCI), provides an excellent model for understanding mechanisms of epigenetic silencing. During development, XCI can take place in either imprinted or random manners. For imprinted XCI, the paternal X chromosome (Xp) is selectively inactivated during preimplantation development. Although imprinted XCI is maintained in the extra-embryonic cell lineage, it is lost in the inner cell mass (ICM) of late blastocysts. At peri-implantation stage, epiblast cells undergo random XCI resulting in the silencing of either Xp or maternal X chromosome (Xm). Previous studies have demonstrated a critical role of Xist, an X-linked long non-coding RNA, in both imprinted and random XCI. The Xist RNA participates in XCI by coating and inactivating X chromosome in cis.

Genomic imprinting allows parent-of-origin specific gene regulation. To selectively silence the Xp during imprinted XCI, the Xist gene is imprinted for silencing in the Xm with a long sought-after, but yet-to-be-identified, mechanism. Previous studies using nuclear transfer approaches have suggested that genomic imprinting of Xist is established during oogenesis, like that of autosomal imprinted genes. In mouse preimplantation embryos and extra-embryonic cells, only the paternal X chromosome (Xp) is inactivated. Central to the imprinted paternal X chromosome inactivation (XCI) is a long non-coding RNA, Xist, which is expressed from Xp and acts in cis to coat and silence the entire Xp. To achieve Xp-specific inactivation, the maternal Xist gene must be silenced, yet the silencing mechanism is not yet clear. As reported herein, the Xist locus is coated with a broad H3K27me3 domain in mouse oocytes, which persists through preimplantation development. Ectopic removal of H3K27me3 induces maternal Xist expression and maternal XCI. Thus, maternal H3K27me3 serves as the imprinting mark of Xist.

In some embodiments, disclosed herein methods related to treating a disorder associated with a H3K27me3-dependent imprinting defect in a subject, comprising administering a pharmaceutical composition comprising a selective H3K27me3 demethylase inhibitor, thereby treating the H3K27me3-dependent imprinting defect.

Therapeutic Methods

Agents that remove H3K27me3 imprinting present in an imprinting control region are useful for preventing or ameliorating a developmental disorder associated with an imprinting control region. Developmental disorders associated with an imprinting control region include, for example, a disorder where one mutant allele (e.g., a paternal allele) is active while a wild-type allele (e.g., a maternal allele) is undesirably silent. Disorders associated with an imprinting control region may be treated by removing H3K27me3 from the undesirably silenced allele, thereby allowing that allele to be expressed.

In one therapeutic approach, an agent that inhibits H3K27me3 demethylase is administered systemically, thereby alleviating the symptoms of the disorder in a subject. The dosage of the administered agent depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgement of the person administering or supervising the administration of the compositions.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

Agents that Modify H3K27me3

Disclosed herein are agents that inhibit histone H3 lysine 27 trimethylation (H3K27me3) thereby activating an H3K27me3 repressed allele. Also disclosed herein are agents (e.g., demethylases, such as KDM6A, KDM6B, and KDM6C) that selectively remove trimethylation at lysine 27 of histone H3 and activate an H3K27me3 repressed allele. Agents that inhibit H3K27me3 are known in the art, and described, for example, in the following patents and patent publications: U.S. Pat. No. 8,895,245 (e.g., Compound, 75, 37, 65, etc.), U.S. Pat. No. 9,688,665, U.S. application Ser. No. 15/101,577, U.S. application Ser. No. 15/211,792, PCT/US2016/065447, PCT/US2016/055554, PCT/US2016/060814; which are incorporated by reference herein. In particular embodiments, the agent is tazemetostat, DZNep, GSK373, GSK126, El1, Epz005687, CPI-169 (See, Morera et al., Clinical Epigenetics 2016 8:57)

In other embodiments, the agents disclosed herein selectively remove trimethylation at lysine 27 of histone H3 and activate an H3K27me3 repressed allele (e.g., KDM6A, KDM6B, or KDM6C). Such demethylases may be expressed as a polynucleotide (e.g., mRNA) in a cell or injected into a cell as a protein.

In accordance with the methods disclosed herein, in therapeutic applications, the dosages of the agents used in accordance with the invention vary depending on the agent, the age, weight, and clinical condition of the of recipient patient, and the experience and judgment of the clinician or practitioner administering the therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the disorder and most preferably causing complete regression of the disorder.

Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is a technique that may be used, for example, for the reproductive cloning of livestock (e.g., cows, horses, sheep, goats) or for therapeutic cloning, in which desired tissues are produced for cell replacement therapy. Unfortunately cloned animals suffer from certain defects arising from improper imprinting, such as a deficiency in trimethylation of lysine 27 on histone H3 protein subunit. This deficiency can be remedied by providing an mRNA encoding an enzyme that carries out the trimethylation event during the SCNT procedure. In one embodiment, an mRNA encoding an enzyme capable of carrying out the trimethylation event (e.g., EZH1, EZH2, PRC2) is injected into the recipient cell or the nuclear donor cell prior to or during the SCNT procedure.

Somatic cell nuclear transfer involves obtaining a nuclear donor cell, then fusing this nuclear donor cell into an enucleated recipient cell, most preferably an enucleated oocyte, to form a nuclear transfer embryo, activating this embryo, and finally culturing the embryo or transferring this embryo into a maternal host. During nuclear transfer a full complement of nuclear DNA from one cell is introduced to an enucleated cell. Nuclear transfer methods are well known to a person of ordinary skill in the art. See, U.S. Pat. No. 4,994,384 to Prather et al., entitled “Multiplying Bovine Embryos,” issued on Feb. 19, 1991; U.S. Pat. No. 5,057,420 to Massey, entitled “Bovine Nuclear Transplantation,” issued on Oct. 15, 1991; U.S. Pat. No. 5,994,619, issued on Nov. 30, 1999 to Stice et al., entitled “Production of Chimeric Bovine or Porcine Animals Using Cultured Inner Cell Mass Cells; U.K. Patents Nos. GB 2,318,578 GB 2,331,751, issued on Jan. 19, 2000 to Campbell et al. and Wilmut et al., respectively, entitled “Quiescent Cell Populations For Nuclear Transfer”; U.S. Pat. No. 6,011,197 to Strelchenko et al., entitled “Method of Cloning Bovines Using Reprogrammed Non-Embryonic Bovine Cells,” issued on Jan. 4, 2000; and in U.S. patent application Ser. No. 09/753,323 entitled “Method of Cloning Porcine Animals (attorney docket number 030653.0026.CIP1, filed Dec. 28, 2000), each of which are hereby incorporated by reference in its entirety including all figures, tables and drawings. Nuclear transfer may be accomplished by using oocytes that are not surrounded by a zona pellucida.

In a nuclear transfer procedure, a nuclear donor cell, or the nucleus thereof, is introduced into a recipient cell. A recipient cell is preferably an oocyte and is preferably enucleated. However, the invention relates in part to nuclear transfer, where a nucleus of an oocyte is not physically extracted from the oocyte. It is possible to establish a nuclear transfer embryo where nuclear DNA from the donor cell is replicated during cellular divisions. See, e.g., Wagoner et al., 1996, “Functional enucleation of bovine oocytes: effects of centrifugation and ultraviolet light,” Theriogenology 46: 279-284. In addition, nuclear transfer may be accomplished by combining one nuclear donor and more than one enucleated oocyte. Also, nuclear transfer may be accomplished by combining one nuclear donor, one or more enucleated oocytes, and the cytoplasm of one or more enucleated oocytes. The resulting combination of a nuclear donor cell and a recipient cell can be referred to as a “hybrid cell.”

The term “nuclear donor” as used herein refers to any cell, or nucleus thereof, having nuclear DNA that can be translocated into an oocyte. A nuclear donor may be a nucleus that has been isolated from a cell. Multiple techniques are available to a person of ordinary skill in the art for isolating a nucleus from a cell and then utilizing the nucleus as a nuclear donor. See, e.g., U.S. Pat. Nos. 4,664,097, 6,011,197, and 6,107,543, each of which is hereby incorporated by reference in its entirety including all figures, tables and drawings. Any type of cell can serve as a nuclear donor. Examples of nuclear donor cells include, but are not limited to, cultured and non-cultured cells isolated from an embryo arising from the union of two gametes in vitro or in vivo; embryonic stem cells (ES cells) arising from cultured embryonic cells (e.g., pre-blastocyst cells and inner cell mass cells); cultured and non-cultured cells arising from inner cell mass cells isolated from embryos; cultured and non-cultured pre-blastocyst cells; cultured and non-cultured fetal cells; cultured and non-cultured adult cells; cultured and non-cultured primordial germ cells; cultured and non-cultured germ cells (e.g., embryonic germ cells); cultured and non-cultured somatic cells isolated from an animal; cultured and non-cultured cumulus cells; cultured and non-cultured amniotic cells; cultured and non-cultured fetal fibroblast cells; cultured and non-cultured genital ridge cells; cultured and non-cultured differentiated cells; cultured and non-cultured cells in a synchronous population; cultured and non-cultured cells in an asynchronous population; cultured and non-cultured serum-starved cells; cultured and non-cultured permanent cells; and cultured and non-cultured totipotent cells. See, e.g., Piedrahita et al., 1998, Biol. Reprod. 58: 1321-1329; Shim et al., 1997, Biol. Reprod. 57: 1089-1095; Tsung et al., 1995, Shih Yen Sheng Wu Hsueh Pao 28: 173-189; and Wheeler, 1994, Reprod. Fertil. Dev. 6: 563-568, each of which is incorporated herein by reference in its entirety including all figures, drawings, and tables. In addition, a nuclear donor may be a cell that was previously frozen or cryopreserved.

Hybrid cells made by the process of nuclear transfer may be used, for example, in reproductive cloning or in regenerative cloning.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES Example 1: Allelic DHSs in Zygotes Mark Promoters that are Primed for Allelic Zygotic Genome Activation

Transcriptional regulatory elements, such as promoters and enhancers, can be mapped by DNase I hyper-sensitivity assay. By using a low-input DNase I-sequencing (liDNase-seq) technique, the transcriptional regulatory landscape of preimplantation embryos were mapped and SNP-based analysis revealed that chromatin accessibility of the two parental alleles is overall comparable except imprinted gene promoters. A similar conclusion was also reached using an assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq). However, the mechanisms underlying parent-of-origin specific chromatin accessibility are unknown.

To comprehensively profile parental allele-specific DHSs in zygotes, paternal and maternal pronuclei from PN5-stage zygotes were isolated and performed liDNase-seq (FIG. 1A, FIG. 2A). Using stringent criteria (FIG. 2B) and excluding data of sex chromosomes, 3,462, 687, and 169 of bi-allelic DHSs were identified, paternal allele-specific DHSs (Ps-DHSs), and maternal allele-specific DHSs (Ms-DHSs), respectively (FIG. 1B, FIG. 2C). The genomic location of allelic DHSs was heavily biased to non-promoter elements when compared to bi-allelic DHSs that were enriched in promoters and CpG islands (FIG. 2D, FIG. 2E). Ps-DHSs include ICRs of known imprinted genes (FIG. 2F). Interestingly, both Ps- and Ms-DHSs also included promoters of genes previously not known to be imprinted (FIG. 2G, FIG. 2H).

Since promoter DHSs can prime gene expression at the next developmental stage, it was explored whether allelic DHSs in zygotes can prime allelic gene expression at zygotic genome activation (ZGA). RNA-seq analysis of 2-cell stage androgenetic (AG) and gynogenetic (GG) embryos, using α-amanitin treatment as a negative control, identified 107 AG- and 14 GG-specific differentially expressed genes (DEGs), including 8 known imprinted genes (FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D).

Integrated analysis of allelic ZGA and allelic promoter DHSs in zygotes revealed that the majority (59% and 79%) of the AG- and GG-specific DEGs were associated with paternal and maternal allele-biased chromatin accessibility, respectively (FIG. 3E, FIG. 3F). Genes showing such a correlation include not only known imprinted genes but also genes not known to be imprinted (FIG. 1C). These results indicated that allelic DHSs in zygotes can mark promoters that are primed for allelic ZGA.

Example 2: DNA Methylation and Allelic DHSs

To understand how allelic DHSs in zygotes were specified, it was examined whether they are inherited from gametes. DHSs of fully-grown oocytes were profiled (FIG. 4A) and analyzed sperm DHSs. Although sperms only have 34 reproducible DHSs, some of them contribute to Ps-DHSs (FIG. 4B). However, most of Ps-DHSs are absent in sperm and oocytes, indicating that they are generated after fertilization (FIG. 4C, FIG. 4D). In contrast, most of Ms-DHSs and bi-allelic DHSs are already present in oocytes (FIG. 4E, FIG. 4F, FIG. 4G, FIG. 4H), indicating that most maternal DHSs are inherited from oocytes.

To determine how the maternal allele at Ps-DHSs remains inaccessible, it was hypothesized that maternal DNA methylation prevents DHS formation. Analysis of a public whole genome bisulfite sequencing (WGBS) dataset of oocytes and sperm revealed that only 17% of Ps-DHSs overlap with oocyte germline differentially methylated regions (gDMRs) (FIG. 5A). Despite extending to a ±100 kb region flanking Ps-DHSs, only additional 21% are found to be associated with oocyte gDMRs (FIG. 5A). Even when the oocyte DNA methylation level alone is considered, 48% of Ps-DHSs are devoid of oocyte DNA methylation (FIG. 5B), indicating the existence of a DNA methylation-independent mechanism that prevents maternal allelic accessibility.

Example 3: Maternal Allelic Protection by H3K27me3

The fact that Polycomb-mediated H3K27me3 can mediate silencing of DNA hypomethylated promoters led to the postulation that H3K27me3 might be responsible for maternal allele inaccessibility. Analyses of public ChIP-seq datasets revealed that the H3K27me3 level in oocytes was much higher than that of sperm at DNA hypomethylated Ps-DHSs, while it was reversed at DNA hypermethylated Ps-DHSs (FIG. 5C, left panel). SNP-tracking analysis revealed that the hypomethylated Ps-DHSs maintain maternal allele-specific H3K27me3 in zygotes (FIG. 5C, right panel), indicating that H3K27me3 may be responsible for maternal allele inaccessibility at DNA hypomethylated regions.

To test this possibility, mRNA encoding an H3K27me3-specific demethylase Kdm6b (Kdm6b^(WT)) with its catalytic mutant (H1390A) (Kdm6b^(MUT)) was injected as a control (FIG. 6A). Similarly, zygotes injected with an H3K9me3-specific demethylase Kdm4d or its catalytic mutant (H189A) were prepared. Both WT and mutant Kdm6b and Kdm4d were expressed at a similar level (FIG. 5D), and Kdm6b^(WT) and Kdm4d^(WT), but not their mutants, significantly reduced H3K27me3 and H3K9me3 levels, respectively (FIG. 5E, FIG. 5F). LiDNase-seq of isolated pronuclei (FIG. 5G, FIG. 5H) revealed that 78 and 150 of the 431 most reliable Ps-DHSs became bi-allelic in Kdm6b^(WT)- and Kdm4d^(WT)-injected zygotes, respectively, while their catalytic mutants had little effect (FIG. 6B, FIG. 6C, FIG. 5I). This result indicated that both maternal H3K27me3 and H3K9me3 were involved in maternal allele inaccessibility. Importantly, Kdm6b-affected Ps-DHSs were largely devoid of oocyte DNA methylation, which was markedly different from Kdm4d-affected Ps-DHSs that locate at DNA hypermethylated regions (FIG. 6D). Consistently, Ps-DHSs specifically affected by Kdm6b, but not Kdm4d, overlap maternal allele-specific H3K27me3 (FIG. 5J). These results indicated that maternal H3K27me3 and H3K9me3 restrict maternal allele accessibility at regions with hypomethylated and hypermethylated DNA, respectively.

Example 4: H3K27me3-Dependent Imprinting

Maternal H3K27me3 serves as a DNA methylation-independent imprinting mark and restricts maternal allele accessibility to mediate H3K27me3-dependent genomic imprinting. To understand to what extent allelic DHSs exist at a later embryonic stage, AG and GG morula embryos were generated (FIG. 7A) and performed liDNase-seq (FIG. 8A). Using the same criteria for allelic DHSs as in zygotes and excluding data of sex chromosomes, 36,569, 247, and 176 of common DHSs were identified, AG-specific DHSs (AG-DHSs), and GG-specific DHSs (GG-DHSs), respectively (FIG. 7B). By SNP-tracking analyses of a public DHS profile of hybrid morula embryos, it was confirmed that AG-DHSs, but not GG-DHSs, recapitulate the corresponding parental allele-specific DHSs (FIG. 8B), indicating that AG-DHSs were physiological. Interestingly, AG-DHSs included almost all known maternally-methylated ICRs (FIG. 8C). This finding raised the possibility that AG-DHSs could serve as indicators of genomic imprinting.

Because both gDMR and maternal H3K27me3 can contribute to maternal allele inaccessibility (FIG. 6 ), their respective contribution to the generation of the 247 AG-DHSs was determined. Analyses of the oocyte DNA methylome identified 183 (74%) AG-DHSs in DNA hypomethylated regions (FIG. 8D). Allelic H3K27me3 enrichment analysis revealed that 112 of the 183 were marked with maternal allele-biased H3K27me3 in inner cell mass (ICM) of blastocysts (FIG. 7C). Of the 112 AG-DHSs, 105 showed maternal allele-specific H3K27me3 enrichment in zygotes [RPM>0.5, FC(Mat/Pat)>4], which suggested that the maternal allele-biased H3K27me3 is inherited from zygotic maternal chromatin. By associating the 105 H3K27me3-marked AG-DHSs with their nearest genes, 76 genes (Table 1, below) were obtained as putative H3K27me3-dependent imprinted genes.

TABLE 1 gene_name gene_chr gene_start gene_end Rbp2 chr9 98390956 98410190 Runx1 chr16 92601711 92826311 Sfmbt2 chr2 10292078 10516880 Slc38a2 chr15 96517823 96530129 Slc38a4 chr15 96825254 96886387 Gramd1b chr9 40105492 40263349 Bbx chr16 50191957 50432502 Sox21 chr14 118632456 118636252 Mbnl2 chr14 120674891 120830920 Prdm11 chr2 92815063 92886301 1700067G17Rik chr1 90912688 90918785 1700095B10Rik chr5 113222312 113230721 Mir692-2b chr4 125181992 125182101 Sh3gl3 chr7 89319728 89455927 Etv6 chr6 133985725 134220165 Tle3 chr9 61220173 61266304 Hunk chr16 90386642 90499798 Gab1 chr8 83288333 83404378 Matn1 chr4 130500300 130511391 Chst1 chr2 92439864 92455409 Clic6 chr16 92498392 92541486 1700110K17Rik chr9 40141057 40150922 Fox11 chr8 123651585 123654544 Mir6241 chr14 118657855 118657958 Otog chr7 53496357 53566804 1700017J07Rik chr2 168803769 168804406 4930404H11Rik chr12 72641594 72657120 Gm5086 chr13 98329955 98353949 Tshz2 chr2 169459146 169714004 Bmp7 chr2 172695189 172765794 G730013B05Rik chr16 50526358 50559572 Rftn1 chr17 50132632 50329822 C430002E04Rik chr3 41291603 41297121 Myoz2 chr3 122709124 122737905 Six3os1 chr17 86001272 86017736 Slc38a1 chr15 96401849 96473344 Rbms1 chr2 60590010 60801261 Flt1 chr5 148373772 148537564 Sall3 chr18 81163113 81183317 Otx2os1 chr14 49288963 49413023 1700006F04Rik chr14 120148449 120150786 2300005B03Rik chr15 74573269 74577117 4931430N09Rik chr6 118830176 118835561 Gas7 chr11 67346500 67502494 Phf17 chr3 41359656 41420786 Igsf21 chr4 139582767 139802726 Otx2 chr14 49277859 49282547 Klhdc7a chr4 139518088 139523941 1700125H03Rik chr8 70892358 70899609 Lpar3 chr3 145883925 145949178 Mir6239 chr14 118352964 118353069 Epas1 chr17 87153204 87232750 Slc6a1 chr6 114232629 114267519 Cdh26 chr2 178165312 178222071 1700025C18Rik chr2 164904193 164916250 Prox1 chr1 191945658 191994559 1700121N20Rik chr12 107680862 107685876 Adamts2 chr11 50415587 50617551 Gadl1 chr9 115818573 115985294 Dnase2b chr3 146244337 146278562 Inhbb chr1 121312042 121318825 E2f3 chr13 29998444 30077932 Ajap1 chr4 152747330 152856939 BC049762 chr11 51067153 51076453 Edn3 chr2 174586274 174609543 Enc1 chr13 98011060 98022995 4930465M20Rik chr12 108961953 108973698 9630028H03Rik chr2 135406266 135408956 Cd44 chr2 102651300 102741822 Epgn chr5 91456543 91464238 Syt13 chr2 92755258 92796208 Myb chr10 20844736 20880790 Lrig3 chr10 125403275 125452415 Fam198b chr3 79689852 79750200 Smoc1 chr12 82127795 82287401 1700084F23Rik chr13 70142928 70167226

To determine if any of the 76 genes are indeed imprinted in preimplantation embryos, RNA-seq analysis was performed for AG and GG morula embryos (FIG. 9A). After confirming AG- or GG-specific expression of known imprinted genes (FIG. 9B), the relative AG/GG expression levels for each candidate was calculated. Among the 76 genes, 28 were expressed in either AG or GG embryos (FPKM>0.5). Interestingly, 27 of the 28 genes exhibited biased (FC>2), and 23 genes exhibited highly biased (FC>8) expression in AG embryos (FIG. 7D, left column). Using a RNA-seq dataset of hybrid IVF morula embryos, it was further confirmed that all 13 SNP-trackable genes exhibit paternal allele-specific expression (FIG. 7D, right columns). Importantly, these genes included Sfmbt2, Gab1, Slc38a4, and Phf17 whose imprinted expression was suggested to be independent of oocyte DNA methylation. These ‘non-canonical’ imprinted genes were coated with oocyte-specific H3K27me3 domains that are retained even in blastocysts (FIG. 9C), which is in contrast to DNA methylation-dependent ‘canonical’ imprinted genes that are devoid of oocyte H3K27me3 (FIG. 9D). Collectively, these results demonstrated that maternal H3K27me3 may serve as a DNA methylation-independent imprinting mark.

To determine whether maternal H3K27me3 was responsible for maternal allele repression of the putative H3K27me3-dependent imprinted genes, Kdm6b^(WT) or Kdm6b^(MUT) mRNAs was injected into 1-cell stage parthenogenetic (PG) embryos (FIG. 10A). After verifying that the injection did not affect embryo development to the morula stage (FIG. 11A), RNA-seq analysis was performed (FIG. 11B). Of the 28 putative imprinted genes expressed in AG morula embryos (FIG. 7D), 16 were significantly derepressed in a catalytic activity-dependent manner, which include all 4 known non-canonical imprinted genes (FIG. 10B). In contrast, canonical imprinted genes were not affected by Kdm6b^(WT) injection (FIG. 11C), demonstrating that H3K27me3 was specifically required for maternal allele repression of the putative H3K27me3-dependent imprinted genes.

To demonstrate that Kdm6b-mediated maternal allele derepression occurs in a physiological context, RNA-seq analysis was performed in IVF-derived hybrid morula embryos that had been injected with Kdm6b^(WT) or Kdm6b^(MUT) mRNA at the 1-cell stage. Of the 28 putative imprinted genes, 17 had sufficient SNP reads, and 16 of them showed paternal allele-biased expression in Kdm6b^(MUT)-injected embryos (FIG. 10C). Notably, the extent of the paternal allelic bias of all these genes became milder in Kdm6b^(WT)-injected embryos, while that of canonical imprinted genes was not affected (FIG. 10C). These data indicated that imprinted expression of these genes depends on maternal H3K27me3.

To determine whether maternal allele derepression couples with gain of maternal chromatin accessibility, liDNase-seq was performed for Kdm6b^(WT)- and Kdm6b^(MUT)-injected PG morula embryos (FIG. 7D). We found that Kdm6b^(WT), but not Kdm6b^(MUT), markedly increased chromatin accessibility in AG-DHSs of putative H3K27me3-dependent imprinted genes, including all 4 non-canonical imprinted genes (FIG. 10D, FIG. 10E and FIG. 11E). In contrast, ICRs of canonical imprinted genes were not affected (FIG. 10D and FIG. 11F, FIG. 11G). These results indicated that maternal H3K27me3 restricts maternal allele accessibility to mediate H3K27me3-dependent genomic imprinting.

Example 5: Imprinting Status in Blastocysts

The imprinting status of putative H3K27me3-dependent imprinted genes was then analyzed in blastocyst embryos by SNP tracking of recently published datasets. Of the 28 genes imprinted in morula embryos (FIG. 7D), 15 had sufficient SNP reads in both reciprocal crosses (FIG. 12A). Among them, 12 (80%) showed paternal allelic expression in both crosses (FIG. 12A), demonstrating that H3K27me3-dependent imprinting was largely maintained in blastocysts.

Since previous studies have indicated that Gab1, Sfmbt2, and Phf17 are imprinted only in extra-embryonic tissues, their imprinting status was examined in ICM. TE and ICM cells were isolated from AG and GG blastocysts and RNA-seq analysis was performed. Marker gene expression confirmed no cross-contamination (FIG. 13A). Of the 28 putative imprinted genes (FIG. 7D), 23 and 24 were expressed in TE and ICM, respectively (RPKM>0.5). Of these, 18 (78%) in TE and 16 (67%) in ICM showed AG-biased expression (FC>2) (FIG. 12B). Notably, 9 genes showed weaker AG-bias in ICM compared to TE (FIG. 12B, arrows), suggesting that H3K27me3-dependent imprinting might start to diminish in ICM.

Example 6: Post-Implantation Imprinting Dynamics

To determine the imprinting status in post-implantation embryos, hybrid E6.5 embryos were dissected into epiblast (EPI), visceral endoderm (VE), and extra-embryonic ectoderm (EXE), and RNA-seq analysis performed (FIG. 13B). Cell identity was confirmed by analyzing cell lineage-specific marker gene expression (FIG. 13C) and identified 17 paternally-expressed genes (PEGs) and 8 maternally-expressed genes (MEGs) in EPI, 19 PEGs and 12 MEGs in both VE and EXE, which included new imprinted genes, such as D7Ertd715e (also known as Snhg14), Smoc1, and Mas1 (FIG. 13D, FIG. 13E).

Among the 76 putative H3K27me3-dependent imprinted genes, 25, 23, and 17 genes had enough SNP reads in both reciprocal crosses in EPI, VE, and EXE, respectively (FIG. 12C). It was found that 1, 3, and 5 genes are paternally expressed in EPI, VE, and EXE, respectively (FIG. 12C, arrowheads). The genes imprinted in EXE included the 4 non-canonical imprinted genes, Gab1, Phf17, Sfmbt2, and Slc38a4, and a new imprinted gene, Smoc1 (FIG. 12C). These results suggested that H3K27me3-dependent imprinting was completely erased in the epiblast with the exception of Slc38a4, but some are maintained in the extra-embryonic cell lineages.

To analyze the imprinting status in E9.5 placentae avoiding possible maternal cell contamination, fetus-derived placental cells were purified from GFP transgenic embryos by FACS-sorting (FIG. 14A) and RNA-seq analysis performed (FIG. 14B). After confirming cell purity by demonstrating comparable total SNP reads from parental alleles (FIG. 14C), 25 PEGs and 21 MEGs were identified, which included new imprinted genes, such as D7Ertd715e, Smoc1, Cbx7 and Thbs2 (FIG. 15A, FIG. 15B). Among the 76 putative H3K27me3-dependent imprinted genes, 27 genes had sufficient SNP reads in both reciprocal crosses (FIG. 12D). Among them, Gab1, Sfmbt2, Slc38a4, and Smoc1 are paternally expressed (FIG. 12D). Imprinting of Phf17 in one cross was weak (FC=1.87) (FIG. 12D), which was consistent with a previous study. Taken together, the data not only identified Smoc1 as a new H3K27me3-dependent imprinted gene, but also suggested that most H3K27me3-dependent imprinted genes are transiently imprinted in preimplantation embryos, while some remain imprinted in the extra-embryonic cell lineage (FIG. 12E).

Since the identification of DNA methylation as a genomic imprinting mark more than 20 years ago, it has been the only known mammalian germline imprinting mark. However, recent studies have identified several imprinted genes capable of maintaining paternal allele-specific expression in the absence of oocyte DNA methylation, suggesting the existence of a DNA methylation-independent imprinting mechanism. As reported herein, these non-canonical imprinted genes harbor high level of oocyte-specific H3K27me3, and loss of H3K27me3 results in loss-of-imprinting. Although previous studies have revealed a link between a repressed allele and repressive histone modifications, including H3K27me3, at certain imprinted loci, the imprinting status of these loci originally depends on gDMRs. Consistently, ectopic removal of H3K27me3 specifically affected non-canonical imprinted genes, indicating that the regulatory mechanism of H3K27me3-dependent imprinting is fundamentally different from that of gDMR-mediated canonical imprinting.

The dynamics of H3K27me3-dependent imprinting is strikingly different from DNA methylation-dependent imprinting which is largely maintained in both embryonic and extra-embryonic lineages. The H3K27me3 imprint mark is likely established during oogenesis and maintained in preimplantation embryos (FIG. 12 e ). While it begins to dilute in ICM and is almost completely lost in the epiblast of E6.5 embryos, it is maintained in some genes at least until E9.5 placenta. Further investigation is warranted to understand why and how these genes are selected to maintain imprinting and why they use H3K27me3, instead of DNA methylation, as an imprinting mark, as well as how cell lineage-specific imprinting is achieved. Furthermore, what other organisms may conserve H3K27me3-dependent genomic imprinting is a fascinating question given that flowering plants also adopt this mechanism.

Example 7: Maternal H3K27me3 Coats Xist

If H3K27me3 serves as the imprinting mark of Xist, it would be present in oocytes, but not sperm. To test this notion, public H3K27me3 ChIP-seq datasets were analyzed, which revealed that the Xist locus was coated with a broad H3K27me3 domain that spans to ˜450 kb including the Xist gene body in oocytes (FIG. 16A). Consistent with previous studies showing that Xist imprinting was established during oocyte growth, the H3K27me3 domain was gradually gained during this period (FIG. 16A). Furthermore, analyses of the oocyte DNaseI-sequencing dataset revealed that the chromatin accessibility of this entire H3K27me3 domain was markedly lower than the surrounding regions (FIG. 16A), suggesting formation of a heterochromatin domain. Analyses of the oocyte DNA methylome revealed that this domain was largely hypomethylated in oocytes (FIG. 16A), which is consistent with a previous report showing that Xist imprinting is independent of oocyte DNA methylation.

To determine whether the maternal allele-specific H3K27me3 observed in oocytes was maintained during preimplantation development, the ChIP-seq datasets of 1-cell, 2-cell, and blastocyst embryos was analyzed. The maternal H3K27me3 domain was found to be maintained throughout preimplantation development (FIG. 16B), and the upstream ˜200 kb region of the H3K27me3 domain, including Xist, maintained the maternal allele-bias of H3K27me3 enrichment in blastocyst embryos (FIG. 16B). In contrast, the downstream ˜250 kb region of the H3K27me3 domain almost loses the allelic difference in blastocysts due to gain of paternal H3K27me3 (FIG. 16B). These data supported a potential role of maternal H3K27me3 in maternal Xist silencing.

Example 8: Maternal H3K27me3 is Responsible for Maternal Xist Silencing

To examine whether H3K27me3 was responsible for maternal Xist silencing, H3K27me3 in zygotes were depleted by injecting mRNA coding an H3K27me3-specific demethylase, Kdm6b^(WT), (FIG. 17A). As a negative control, zygotes injected with its catalytic mutant, Kdm6b^(MUT), harboring a point mutation at the catalytic domain were prepared. This approach allowed efficient reduction of H3K27me3 in zygotes in a catalytic activity-dependent manner (FIG. 19A, FIG. 19B). To visualize Xist RNA expression, RNA fluorescent in situ hybridization (FISH) analysis was performed at the morula stage. To distinguish between male and female embryos, X chromosomes were simultaneously labeled by performing DNA FISH with a probe for an X chromosome locus harboring Rnf12. As such, one and two DNA FISH signals in a blastomere can distinguish male and female embryos, respectively. As expected, RNA/DNA FISH analysis revealed that the majority of Kdm6b^(MUT)-injected females showed one RNA cloud, whereas males showed no RNA cloud signal (FIG. 17B, FIG. 17C). In contrast, the majority of Kdm6b^(WT)-injected males and females showed one and two Xist RNA clouds, respectively (FIG. 17B, FIG. 17C, FIG. 17D), demonstrating that H3K27me3 is responsible for repression of the maternal Xist in preimplantation embryos.

Next, it was whether ectopic loss of H3K9me3 in normal, bi-parental, embryos leads to maternal Xist derepression, as in the case of PG embryos. To this end, mRNA coding Kdm4d in zygotes was injected, which efficiently reduced H3K9me3 in a catalytic activity-dependent manner (FIG. 20A, FIG. 20B). RNA/DNA FISH analysis at the morula stage embryos revealed that expression of Kdm4d^(WT) did not induce maternal Xist expression in either male or female embryos (FIG. 20C, FIG. 20D, FIG. 20E), suggesting that H3K9me3 did not play a major role in maternal Xist repression under physiological conditions.

Example 9: Loss of H3K27me3 Induces Maternal XCI

To determine whether maternal Xist expression led to maternal XCI in Kdm6b^(WT)-injected embryos, RNA-seq analysis was performed on early blastocyst embryos. To distinguish between parental alleles, hybrid strain embryos derived from BDF1 oocytes fertilized with PWK sperm were prepared. The biological duplicates of RNA-seq datasets were highly reproducible (FIG. 21 ). Analysis of single nucleotide polymorphism (SNP) information revealed that the expression level of the maternal allele of X-linked genes, but not those of autosomal genes, was significantly downregulated in Kdm6b^(WT)-injected embryos (FIG. 18A). A closer examination of individual SNP-tracked X-linked genes confirmed that most genes were downregulated (FIG. 18B). Furthermore, genes known to escape imprinted XCI (called ‘escapees’) escaped from the maternal XCI (FIG. 18C). These data further support the responsibility of H3K27me3 for maternal Xist silencing to prevent maternal XCI.

The results described herein above, were obtained using the following methods and materials.

Isolation of Maternal and Paternal Pronuclei from PN5 Stage Zygotes

All animal studies were performed in accordance with guidelines of the Institutional Animal Care and Use Committee at Harvard Medical School. MII-stage oocytes were collected from 8 week-old B6D2F1/J (BDF1) females superovulated by injecting 7.5 I.U. of PMSG (Millipore) and hCG (Millipore). For in vitro fertilization (IVF), MII oocytes were inseminated with activated spermatozoa obtained from the caudal epididymis of adult BDF1 male mice in HTF medium supplemented with 10 mg/ml bovine serum albumin (BSA; Sigma-Aldrich). Spermatozoa capacitation was attained by 1 h incubation in the HTF medium. Zygotes were cultured in a humidified atmosphere with 5% CO₂/95% air at 37.8° C. At 10 hours post-fertilization (hpf), zygotes were transferred into M2 media containing 10 μg/ml cytochalasin B (Sigma-Aldrich). Zona pellucidae were cut by a Piezo impact-driven micromanipulator (Prime Tech Ltd., Ibaraki, Japan) and the pronuclei were isolated from the zygotes. At 12 hpf (PN5-stage), isolated pronuclei were washed with 0.2% BSA/PBS, transferred into Eppendorf LoBind 1.5 ml tubes, and placed on ice until DNase I treatment. For each experiment, 150-200 pronuclei were collected and prepared for liDNase-seq. The parental pronuclei were distinguished by (1) the distance from the second polar body and (2) the size of the pronucleus.

Preparation of Androgenetic (AG) and Gynogenetic (GG) Embryos

MII oocytes were collected from 8 week-old superovulated BDF1 females and inseminated with BDF1 sperm. At 7 hpf, zygotes were transferred into M2 media containing 5 μg/ml cytochalasin B, and parental pronuclei were exchanged by using a Piezo impact-driven micromanipulator. The sendai virus (HVJ, Cosmo-bio) was used for fusing karyoplasts with cytoplasms as previously described. After reconstruction, embryos were cultured in KSOM.

When collecting embryos for RNA-seq or/and liDNase-seq, we removed zona pellucida (ZP) by a brief exposure to Acid tyrode's solution (Sigma-Aldrich), then the embryos were washed with M2 media, and then 0.2% BSA/PBS. For liDNase-seq, 10 morula embryos were transferred into an Eppendorf LoBind 1.5 ml tube, and placed on ice until DNase I treatment. For RNA-seq, seven to ten embryos were transferred into a thin-walled RNase-free PCR tubes (Ambion). The 2-cell and morula embryos were collected at 30 and 78 hpf, respectively. When preparing α-amanitin treated 2-cell embryos, 5 hpf zygotes were transferred into KSOM containing 25 μg/ml α-amanitin (Sigma-Aldrich) and cultured in the presence of α-amanitin until collection (30 hpf). ICM and TE were isolated. Briefly, AG and GG embryos at 120 hpi were treated with Acid tyrode's solution to remove ZP. After being washed in M2 media, the embryos were incubated in KSOM containing rabbit anti-mouse lymphocyte serum (Cedarlane, 1:8 dilution) for 45 min at 37° C. After being washed in M2 media, they were transferred into KSOM containing guinea pig complement (MP Biomedicals, 1:3.3 dilution). After incubation for 30 min at 37° C., lysed TE cells were removed by pipetting with a glass capillary. The remaining ICM clumps were incubated in 0.25% Trypsin/EDTA (Thermo Fisher, 25200) for 10 min at 37° C., and then dissociated into single cells to avoid contamination of lysed TE cells. 100-200 cells were collected for RNA-seq.

Isolation of GV Nuclei from Fully-Grown Oocytes

Fully-grown GV-stage oocytes were obtained from 3-week-old BDF1 mice 44-48 h after injection with 5 LU. PMSG. The ovaries were transferred to M2 media. The ovarian follicles were punctured with a 30-gauge needle, and the cumulus cells were gently removed from the cumulus-oocyte complexes using a narrow-bore glass pipette. The oocytes were then transferred into α-MEM (Life technologies, 12571-063) supplemented with 5% Fetal Bovine Serum (FBS) (Sigma-Aldrich, F0926), 10 ng/ml Epidermal Growth Factor (Sigma-Aldrich, E4127), and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich). One hour after collection, GV oocytes exhibiting visible perivitelline spaces, which have the surrounding-nucleolus (SN)-type chromatin, were culled. They were then incubated in M2 media containing 10 μg/ml cytochalasin B, 0.1 μg/ml colcemid (Sigma-Aldrich), and 0.2 mM IBMX for 15 min. Then, GV nuclei were isolated by using a Piezo-driven micromanipulator. After washing with 0.2% BSA/PBS, the GV nuclei were transferred into an Eppendorf LoBind 1.5 ml tube. For each experiment, 115-150 GV nuclei were collected for liDNase-seq.

Dissection of E6.5 Embryos and FACS Sorting of GFP-Positive E9.5 Placental Cells

To obtain C57BL6(B6)/PWK hybrid embryos, a natural mating scheme was used. To obtain PWK/B6 hybrid embryos, in vitro fertilization of PWK oocytes with B6 sperm was used, and the 2-cell embryos were transferred into surrogate ICR strain mothers. Dissection of E6.5 embryos into EPI, EXE, and VE was performed. To collect E9.5 placental cells, the B6^(GFP) mice from Jackson laboratory were purchased [C57BL/6-Tg(CAG-EGFP)131Osb/LeySopJ, Stock number 006567]. MII oocytes and sperms were collected from superovulated 8-week old B6^(GFP) or PWK mice. After in vitro fertilization, the 2-cell embryos were transferred into surrogate ICR strain mothers. At E9.5, placentae were harvested, cut into ˜0.5 mm pieces, transferred into 50 ml tubes, and treated with 2 ml of 0.25% Trypsin-EDTA (Thermo Fisher Scientific, 25200) at 30° C. for 15 min in a shaker at 200 rpm to dissociate placental cells. Trypsin treatment was stopped by the addition of 2 ml DMEM containing 10% FBS. After pipetting, the tubes were centrifuged and the pelleted cells were washed with 0.2% BSA/PBS three times. DAPI was added at the final concentration of 1 μM in the final cell suspension. The GFP-positive cells were sorted using a BD FACSaria machine (BD Biosciences) with DAPI positive cells excluded as dead cells. Approximately 10,000-20,000 GFP-positive cells were collected from each placenta, which corresponded to 40-60% of total placental cells.

Plasmid Construction and mRNA Preparation

To generate the Kdm6b^(WT) construct, the cDNA encoding the carboxyl-terminal part containing the catalytic domain (amino acid 1025-End) was amplified. The PCR amplicon was cloned between a Flag tag and poly(A) of the pcDNA3.1-Flag-poly(A)83 plasmid. The H1390A Kdm6b^(MUT) construct were generated by using PrimeSTAR mutagenesis (TAKARA). Primers used for the mutagenesis are 5′-CCAGGCgctCAAGAGAATAACAATTTCTGCTCAGTCAACATCAAC-3′ (SEQ ID NO: 16) and 5′-CTCTTGagcGCCTGGCGTTCGGCTGCCAGGGACCTTCATG-3′ (SEQ ID NO: 17). All constructs were verified by DNA sequencing. The plasmids for wild-type and H189A mutant Kdm4d were previously described.

After linearization by a restriction enzyme, the construct was purified with phenol-chloroform extraction. mRNA was synthesized by in vitro transcription using a mMESSAGE mMACHINE T7 Ultra Kit (Life technologies) according to manufacturer's instructions. The synthesized mRNA was purified by lithium chloride precipitation and diluted with nuclease-free water. mRNA aliquots were stored in −80° C. until use.

mRNA Injection

MII oocytes were collected from superovulated 8 week-old BDF1 females and inseminated with BDF1 sperm. At 2.5 hpf, fertilized oocytes were transferred into M2 media and mRNA was injected using a Piezo impact-driven micromanipulator. mRNA injection was completed by 4 hpf. The mRNA concentrations of Kdm6b^(WT) and Kdm6b^(MUT) were 1.8 μg/μl, and those of Kdm4d^(WT) and Kdm4d^(MUT) were 1.5 μg/μl. When preparing Kdm6b-injected PG embryos, MII oocytes were chemically activated by treating with 3 mM SrCl₂ in Ca²⁺-free KSOM containing 5 μg/ml cytochalasin B. At 4 hrs post-activation (hpa), the embryos were washed with KSOM. At 5 hpa, they were injected with mRNA.

Whole Mount Immunostaining

Zygotes were fixed in 3.7% paraformaldehyde (PFA) in PBS containing 0.2% Triton for 20 min. After 4× washes with PBS containing 10 mg/ml BSA (PBS/BSA), zygotes were treated with primary antibodies at 4° C. overnight. The primary antibodies used in this study were mouse-anti-H3K27me3 (1/500, Active Motif, 61017), rabbit anti-H3K9me3 (1/500, Millipore, 07-442), and rabbit anti-FLAG (1/2000, Sigma-Aldrich, F7524). After 3× washes with PBS/BSA, samples were incubated with a 1:250 dilution of fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson Immuno-Research) or Alexa Flour 568 donkey anti-rabbit IgG (Life technologies) for 1 h. The zygotes were then mounted on a glass slide in Vectashield anti-bleaching solution with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Fluorescence was detected under a laser-scanning confocal microscope with a spinning disk (CSU-10, Yokogawa) and an EM-CCD camera (ImagEM, Hamamatsu) or Zeiss LSM800.

All images were acquired and analyzed using the Axiovision software (Carl Zeiss). The fluorescent signal intensity was quantified with the Axiovision software. Briefly, the signal intensity within the maternal pronuclei was determined, and the cytoplasmic signal was subtracted as background. Then, the averaged signal intensity of the no-injection control zygotes was set as 1.0.

Low-Input DNase-Seq

Low-input DNase-seq libraries were prepared as previously described with minor modifications. Embryos or nuclei collected in 1.5 ml tubes were resuspended in 36 μl lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Triton X-100) and incubated on ice for 5 min. DNase I (10 U/μl, Roche) was added to the final concentration of 80 U/ml (for the GV nucleus sample) or 40 U/ml (for all the other samples) and incubated at 37° C. for exactly 5 min. The reaction was stopped by adding 80 μl Stop Buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.15% SDS, 10 mM EDTA) containing 2 μl Proteinase K (20 mg/ml, Life technologies). Then 20 ng of a circular carrier DNA [a pure plasmid DNA without any mammalian genes purified with 0.5× Beckman SPRIselect beads (Beckman Coulter) to remove small DNA fragments] was added. The mixture was incubated at 50° C. for 1 hr, then DNA was purified by extraction with phenol-chloroform and precipitated by ethanol in the presence of linear acrylamide (Life technologies) overnight at −20° C. Precipitated DNA was resuspended in 50 μl TE (2.5 mM Tris, pH 7.6, 0.05 mM EDTA), and the entire volume was used for sequencing library construction.

Sequencing library was prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufactures' instruction with the exception that the adaptor ligation was performed with 0.03 μM adaptor in the ligation reaction for 30 minutes at 20° C. and that PCR amplification was performed using Kapa Hifi hotstart readymix (Kapa Biosystems) for 8-cycles. The PCR products were purified with ×1.3 volume of SPRIselect beads (Beckman Coulter) and then size selected with ×0.65 volume followed by ×0.7 volume of SPRIselect beads. The sample was eluted in 24 p 1 TE. The number of cycles needed for the second PCR amplification was determined by qPCR using 1 μl of the 1:1,000 diluted samples. The remaining 23 μl of the samples was then amplified with Kapa Hifi hotstart readymix (we used 7 cycles for all samples in this study). The PCR product was purified with ×1.3 volume of SPRIselect beads and then size selected with ×0.65 volume followed by ×0.7 volume of SPRIselect beads. The DNA was eluted in 30 μl of TE and quantified by Qubit dsDNA HS assay kit (Thermo Fisher Scientific, Q32854) and Agilent high sensitivity assay kit (Agilent Technologies). The libraries were sequenced on a Hiseq2500 with single-end 100 bp reads (Illumina).

RNA-Sequencing

RNA-seq libraries were prepared as previously described. Briefly, reverse transcription and cDNA amplification were performed using whole embryo lysates with SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 634890). When processing 2-cell AG, GG and α-amanitin-treated IVF embryo samples, 1 μl of 1:40,000 diluted ERCC (External RNA Controls Consortium) standard RNA (Life technologies) was added to each of the tubes at the step of cell lysis. cDNAs were then fragmented using the Covaris M220 sonicator (Covaris) with microTUBE-50 (Covaris) into average 150-160 bp fragments. The fragmented cDNAs were end-repaired, adaptor ligated and amplified using NEBNext Ultra DNA Library Prep Kit for Illumina according to the manufacturer's instruction (New England Biolabs). Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina).

liDNase-Seq Data Analysis

Reads of liDNase-seq data were firstly trimmed of low quality and adapter with trim_galore, and then mapped to the mouse genome (mm9) using Bowtie v0.12.9. ‘−m 1’ parameter to keep unique mapping hits. The reads with mapping quality (MAPQ)≤10 or redundant reads that mapped to the same location with the same orientation were removed with SAMtools. The DHS peaks in liDNase-seq data were identified by Hotspot program with FDR<=0.01. The DHS peaks from all 33 libraries were merged using ‘bedtools merge’ from bedtools. The number of reads in each DHS for each library was calculated using ‘multiBamSummary’ from deepTools and normalized to the total number of mapped reads and to the length of DHS (possibility of a tag located on a position per 1 kb per million mapped reads). Reads of sex chromosomes were removed because the number of sex chromosomes is different between the parental pronuclei and between androgenetic and gynogenetic embryos. The Pearson correlation coefficient (r) of tag densities at genome-wide DHSs was calculated to measure the correlation between replicates. For identification of parental allele-specific DHSs in zygotes and morula embryos, we used a stringent cutoff (RPKM mean>2, RPKM>1 in all replicates in a biased allele, and mean value fold change larger than 4 between the two alleles). The 431 most reliable Ps-DHSs were identified by applying an additional criterion ‘RPKM>1 in all replicates of paternal PNs of microinjected zygotes’ to Ps-DHSs. The RefSeq gene assembly (mm9) from the UCSC Genome Browser database and CGIs previously defined were used as genomic feature distribution analysis in FIGS. 2 d and 2 e.

RNA-Seq Data Analysis

We constructed a custom reference sequence combining mouse genome (mm9) with the ERCC control. Reads of RNA-seq were mapped to the reference genome with TopHat v2.0.6 or STAR (github.com/alexdobin/STAR). All programs were run with default parameters unless otherwise specified. Uniquely mapped reads were subsequently assembled into transcripts guided by the reference annotation (UCSC gene models) with featureCounts from subread-v1.5.1. For all 2-cell RNA-seq libraries, library size factors were estimated with ‘estimateSizeFactors’ function form R package DESeq only using ERCC read counts. After the library size was normalized, the expression level of each gene was quantified with normalized FPKM (fragments per kilobase of exon per million mapped fragments). The Pearson correlation coefficient (r) of gene expression level was calculated to indicate the correlation between duplicates. For identification of newly synthesized transcripts at the 2-cell stage, we firstly filtered out statistically non-significant genes between AG or GG and α-amanitin treated 2-cell embryo. To this end, adjusted P value was calculated with ‘nbinomTest’ function form R package DESeq using a negative binomial model, and only genes with FDR<0.05 were selected. We then applied additional cutoffs [Mean FPKM (AG or GG)>2 and fold-change (FC) (AG/Ama or GG/Ama)>2]. As a result, 4,381 and 3,916 genes were identified as newly synthesized genes in AG and GG 2-cell embryos, respectively. For identifying AG- and GG-specific DEGs in 2-cell embryos, the gene expression level (FPKM) of each gene in α-amanitin 2-cell embryos was subtracted from that of AG and GG embryos. Genes showing FC (AG/GG or GG/AG)>10 were identified as DEGs.

WGBS and H3K27me3 ChIP-Seq Data Analyses

The DNA methylation level at DHSs was calculated using methpipe v3.4.2. When calculating the DNA methylation level at each DHS, to get enough coverage of WGBS reads, we extended each DHS to both up and downstream 2 kb to include more nearby CpG sites. The oocyte-methylated gDMR was defined by >80% methylation in oocytes and <20% in sperm. For FIG. 5 a , “bedtools makewindows” were used to generate a set of non-overlapped 1 kb bins for the ±100 kb flanking region of Ps-DHSs. For H3K27me3 ChIP-seq analysis, Bed files were downloaded from Zheng et al., 2016 and converted to the bigWig format using ‘bedClip’ and ‘bedGraphToBigWig’ from UCSC Genome Browser database. ‘multiBigwigSummary’ from deepTools was used to compute H3K27me3 signal over the DHS and surrounding region.

Statistical Analyses and Data Visualization

Statistical analyses were implemented with R (www.r-project.org/). Pearson's r coefficient was calculated using the ‘cor’ function with default parameters. FIG. 6 b and FIG. 10 d were generated with R function ‘heatmap.2’. FIG. 7 d , FIG. 10 c , FIG. 12 a-d were generated with R function ‘pheatmap’. FIG. 1 b and FIG. 7 b were generated using ‘computeMatrix’ and ‘plotHeatmap’ function in deepTools. Position-wise coverage of the genome by sequencing reads was determined by normalizing to the total unique mapped reads in the library using macs2 v2.1.0 and visualized as custom tracks in the IGV genome browser.

Known Imprinting Gene Information

Known imprinting information was downloaded from Error! Hyperlink reference not valid.www.geneimprint.com/site/genes-by-species.Mus+musculus.

Code Availability

A customized pipeline was used to split the hybrid RNA-seq data to their parental origin based on SNP information. The code can be found at github.com/lanjiangboston/UniversalSNPsplit.

Data Availability Statement

All the liDNase-seq and RNA-seq datasets generated in this study were deposited at GEO database under accession number GSE92605. Sperm liDNase-seq datasets were from a previously publication (GSE76642). WGBS datasets for sperm and GV oocytes were downloaded from www.nodai-genome.org/mouse.html?lang=en. H3K27me3 ChIP-seq datasets of sperm, MII oocytes, and SNP-tracked maternal and paternal alleles of 1-cell embryos were downloaded from a previous publication (GSE76687).

Collection of Mouse Preimplantation Embryos

All animal studies were performed in accordance with guidelines of the Institutional Animal Care and Use Committee at Harvard Medical School. MII-stage oocytes were collected from 8 week-old B6D2F1/J (BDF1) females superovulated by injecting 7.5 I.U. of PMSG (Millipore) and hCG (Millipore). For in vitro fertilization (IVF), MII oocytes were inseminated with activated spermatozoa obtained from the caudal epididymis of adult BDF1 or PWK (Jackson Laboratory, 003715) males in HTF medium supplemented with 10 mg/ml bovine serum albumin (BSA; Sigma-Aldrich). Spermatozoa capacitation was attained by 1 h incubation in the HTF medium. Zygotes were transferred to KSOM and cultured in a humidified atmosphere with 5% CO₂/95% air at 37.8° C.

mRNA Injection

At 4 hrs post-fertilization (hpf), zygotes were transferred into M2 media and mRNA was injected using a Piezo impact-driven micromanipulator (Prime Tech Ltd., Ibaraki, Japan). The construction and preparation of mRNA were described above. The concentrations of injected mRNA of Kdm6b^(WT) and Kdm6b^(MUT) were 1.8 μg/μl, and those of Kdm4d^(WT) and Kdm4d^(MUT) were 1.5 μg/μl.

Probe for Fluorescent In Situ Hybridization

A probe for Xist RNA was prepared by using Nick translation reagent kit (Abbott Molecular, 07J00-001) with Cy3-dCTP (GE healthcare, PA53021), according to the manufacturer's instruction. The template DNA used for the probe preparation was a plasmid coding the full-length mouse Xist gene, a gift from Rudolf Jaenisch (pCMV-Xist-PA, 26760) (Wutz and Jaenisch, 2000). A probe for DNA FISH was prepared using the same kit with Green-dUTP (Abbott Molecular, 02N32-050). The template DNA was a BAC clone containing the Rnf12 locus (RP23-36C20). The fluorescent probes were ethanol precipitated with 5 μg Cot-1 DNA (Life technologies), 5 μg herring sperm DNA (Thermo Fisher Scientific), and 2.5 μg yeast tRNA (Thermo Fisher Scientific, AM7119), and then dissolved with 20 μl formamide (Thermo Fisher Scientific, 17899). The probes were stored at 4° C. Before being used, the probes (0.75 μl each) were mixed with 0.75 μl Cot-1 DNA, which had been ethanol precipitated and dissolved in formamide, and 2.25 μl of 4×SSC/20% Dextran (Millipore S4030). The probe mixtures were heated at 80° C. for 30 min and then transferred to a 37° C. incubator (‘pre-annealed probes’).

Whole Mount RNA/DNA Fluorescent In Situ Hybridization

Morula embryos were fixed at 78 hpf in 2% paraformaldehyde (PFA) in PBS containing 0.5% Triton X-100 for 20 min at room temperature. After 3× washes with PBS containing 1 mg/ml BSA (PBS/BSA), embryos were treated with 0.1 N HCl containing 0.02% Triton X-100 for 15 min at 4° C. After 3× washes with 2×SSC containing 0.1% BSA, embryos were incubated in 15 μl of 10% formamide/2×SSC in a glass dish (Electron Microscopy Science, 705430-30). All embryos were sunk and attached to the bottom of the glass dish by gentle pipetting. After 5 min, 15 μl of 30% formamide/2×SSC was added. After 5 min, 90 μl of 60% formamide/2×SSC was added to make the final formamide concentration 50%, and embryos were incubated for additional 30 min at room temperature. The formamide solution containing embryos were covered with mineral oil. The samples were heated at 80° C. for 30 min, and then transferred to a 37° C. incubator for at least 30 min. The embryos were picked in a glass pipette, transferred into 4.5 μl of ‘pre-annealed probes’ covered with mineral oil on another glass dish, and incubated in 37° C. for at least 24 hrs. Embryos were washed with pre-warmed (42° C.) 2×SSC containing 0.1% BSA and left in the last drop for 30 min. After 3× wash with 1% BSA/PBS, they were mounted on a glass slide in Vectashield anti-bleaching solution with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA). Fluorescence was detected under a laser-scanning confocal microscope Zeiss LSM800.

Whole Mount Immunostaining

The procedure of immunostaining and quantification was described above.

Computational Identification of Maternal Allele-Biased H3K27me3

The bed files including RPKM values in 100 bp bins for H3K27me3 ChIP-seq in inner cell mass (ICM) were downloaded from GEO under the number GSE76687. Bed files labeled maternal or paternal containing RPKM values for two parental alleles and allelic reads were normalized to total reads number. ‘bedtools makewindows’ was used to generate 1000 bp bins for mm9 genome, then RPKM value for each bin was calculated by ‘bedtools map’. All the bins are classified to three categories of no signal, biallelic, maternal bias using a signal cutoff of 1 and a fold change cutoff of 4. A sliding window approach was used to identify windows containing maternal biased H3K27me3 bins with criteria of the window size of 20 kb, the minimum bin number of 3 and the percentage of maternal biased H3K27me3 bins larger than 50%. Overlapped windows were merged with “bedtools merge”. A total of 5986 windows were identified in the genome.

RNA-Sequencing

RNA-seq libraries were prepared as described above with minor modifications. Briefly, reverse transcription and cDNA amplification were performed using whole embryo lysates with SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech, 634890). cDNAs were then fragmented using Tagmentation with Nextera XT DNA library prep kit (Illumina). The fragmented cDNAs were amplified using Nextera PCR master mix according to the manufacturer's instruction. Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina).

RNA-Seq Data Analysis

Reads of RNA-seq were mapped to the reference genome with STAR (github.com/alexdobin/STAR). All programs were run with default parameters unless otherwise specified. Uniquely mapped reads were subsequently assembled into transcripts guided by the reference annotation (UCSC gene models) with featureCounts from subread-v1.5.1. After the library size was normalized, the expression level of each gene was quantified with normalized FPKM (fragments per kilobase of exon per million mapped fragments). The Pearson correlation coefficient (r) of gene expression level was calculated to indicate the correlation between duplicates.

Statistical analyses were implemented with R (www.r-project.org/). Pearson's r coefficient was calculated using the ‘cor’ function with default parameters. FIG. 18A was generated with R function ‘boxplot’. FIG. 18B was generated with R function ‘plot’.

Code Availability

A customized pipeline was used to split the hybrid RNA-seq data to their parental origin based on SNP information. The code can be found at github.com/lanjiangboston/UniversalSNPsplit.

Data Availability

RNA-seq datasets generated in this study were deposited at GEO database under accession number GSEXXXXX. The WGBS dataset for GV oocytes was downloaded from www.nodai-genome.org/mouse.html?lang=en. WGBS reads from same 100 bp bins were pooled together to calculate the average methylation level and minimal coverage of 10 reads was required. H3K27me3 ChIP-seq datasets of sperm, MII oocytes, and SNP-tracked maternal and paternal alleles of 1-cell, 2-cell, and inner cell mass of blastocyst embryos were downloaded from a previous study (GSE76687). The oocyte DNaseI-seq datasets were from above (GSE92605).

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. 

What is claimed is:
 1. A method of treating a subject having a disorder associated with histone 3 lysine 27 trimethylation (H3K27me3)-dependent imprinting, the method comprising administering to the subject an agent that inhibits H3K27me3 or an agent that selectively removes trimethylation at lysine 27 of histone 3, thereby treating the disorder.
 2. The method of claim 1, wherein the disorder is associated with a mutation in a gene of Table 1 or in a gene selected from the group consisting of Adamts2, Bbx, BC049762, Bmp7, C430002E04Rik, E2f3, Enc1, Epas1, Etv6, Fam198b, G730013B05Rik, Gab1, Gramd1b, Mbnl2, Otx2, Otx2os1, Phf17, Rbms1, Rbp2, Runx1, Sfmbt2, Sh3gl3, Slc38a1, Slc38a2, Slc38a4, Smoc1, Sox21, and Tle3.
 3. The method of claim 1, wherein the disorder is associated with a mutation in a gene selected from the group consisting of Sfmbt2, Bbx, C430002E04Rik, Phf17, Slc38a4, Gramd1b, Tle3, E2f3, Smoc1, Sox21, Slc38a1, Runx1, Bmp7, Rnc1, Fam198b, Rbms1, Zrsr1, Impact, and Fkbp6.
 4. The method of claim 1, wherein the disorder is associated with a mutation in a gene selected from the group consisting of Sfmbt2, Gab1, Slc38a4, and Phf17.
 5. The method of claim 1, wherein the disorder is associated with a mutation in a gene selected from the group consisting of Ev6, 17001125H03Rik, Smoc1, and Bmp7.
 6. The method of claim 1, wherein the disorder is associated with a mutation in a gene selected from the group consisting of Gab1, Phf17, Sfmbt2, Slc38a4, or Smoc1.
 7. The method of claim 1, wherein the disorder is microphthalmia with limb anomalies (MLA) associated with a mutation in Smoc1.
 8. The method of claim 1, wherein the disorder is associated with limb development associated with a mutation in Smoc1.
 9. The method of claim 1, wherein the disorder is associated with a placental defect associated with a mutation in Gab1 or Sfmbt2.
 10. The method of claim 1, wherein a cell of the subject is contacted with the agent.
 11. The method of claim 10, wherein the agent is an H3K27me3-specific demethylase.
 12. The method of claim 11, wherein the agent is lysine-specific demethylase 6A (KDM6A), lysine-specific demethylase 6B (KDM6B), or lysine-specific demethylase 6C (KDM6C).
 13. The method of claim 10, wherein the agent is an inhibitor of histone 3 lysine 27 trimethylation (H3K27me3).
 14. The method of claim 13, wherein the agent is a small molecule compound, a polypeptide, or a polynucleotide.
 15. The method of claim 14, wherein the agent is selected from the group consisting of tazemetostat, DZNep, GSK373, GSK126, El1, Epz005687, and CPI-169.
 16. The method of claim 10, wherein the cell is contacted in vitro or in vivo.
 17. The method of claim 10, wherein the cell is present in a mammalian subject undergoing pre- or post-natal development. 